Rapid read-out biological indicator

ABSTRACT

A rapid method of determining the efficacy of a sterilization cycle, and an indicator adapted to perform such method, comprising subjecting to the sterilization cycle a source of active enzyme having activity which correlates with the viability of a microorganism commonly used to monitor sterilization, and incubating the enzyme source, following the completion of the sterilization cycle, with an effective amount of a substrate system capable of reacting with any residual active enzyme to produce a detectable enzyme-modified product.

This application is a continuation application of U.S. Ser. No.08/106,816, filed Aug. 3, 1993, allowed and to be issued as U.S. Pat.No. 5,418,167, May 23, 1995, which is a divisional of U.S. Ser. No.07/748,327, filed Aug. 21, 1991, issued as U.S. Pat. No. 5,252,484, Oct.12, 1993, which is a divisional of U.S. Ser. No. 07/277,305, filed Nov.29, 1988, issued as U.S. Pat. No. 5,073,488, Dec. 17, 1991.

FIELD OF THE INVENTION

The present invention relates to a rapid method for determining theefficacy of a sterilization cycle. In particular, the present inventionemploys an enzyme whose activity can be correlated with the viability ofat least one microorganism commonly used to monitor sterilizationefficacy, hereinafter referred to as a “test microorganism”. The enzyme,following a sterilization cycle which is sublethal to the testmicroorganism, remains sufficiently active to react with an enzymesubstrate in a relatively short period of time, e.g., normally eighthours or less. However, the enzyme is inactivated or appreciably reducedin activity following a sterilization cycle which is lethal to the testmicroorganism. The invention further relates to biological sterilityindicators which include such an enzyme.

BACKGROUND OF THE INVENTION

Biological indicators and chemical indicators used to determine theefficacy of sterilization are well known in the art. In conventionalbiological indicators, a test organism which is many times moreresistant to the sterilization process employed than most organismswhich would be present by natural contamination, is coated on a carrierand placed in a sterilizer along with the articles to be sterilized.After completion of the sterilization cycle, the carrier is incubated innutrient medium to determine whether any of the test organism survivedthe sterilization procedure. Growth of a detectable number of organismsnormally takes a minimum of twenty-four hours. During this period, thesupposedly sterilized articles should be quarantined.

In frequent practice, however, the hospital has neither the space forproper quarantining of the supposedly sterilized articles, nor asufficient number of the articles themselves to permit actualquarantining. As a result, the supposedly sterilized articles are placedback into stock on the assumption that sterilization was proper and willbe confirmed by a subsequent report from the laboratory.

Commercially available chemical indicators utilize chemicals whichindicate sterility by color changes, or change from solid to liquidstate. The advantage to such chemical indicators is that the results areknown by the end of the sterilization cycle. However, those resultsindicate only, as in the device described in U.S. Pat. No. 4,448,548,that a particular temperature has been reached for a certain period oftime; or, as in U.S. Pat. No. 4,348,209, that ethylene oxide gas waspresent. These devices do not indicate whether all conditions necessaryto inactivate the test organism have been achieved. Only the livingorganism can sense the true relationships of physical and chemicalparameters necessary to affect sterilization. Therefore, it isrecognized in the art of sterilization that biological tests are themost accurate sterility tests.

There remains a need for a sterility indicator which will provide rapidresults, yet provide a high level of confidence that all parameters,necessary to achieve sterilization, including the interrelatedparameters of time, temperature and concentrations of moisture,chemicals or radiation dose, have been reached.

SUMMARY OF THE INVENTION

The present invention provides a method of determining the efficacy of asterilization cycle, whether the sterilizing media be steam, dry heat,radiation, ethylene oxide, or other gaseous or liquid agents, whichcombines the reliability of the conventional biological indicators witha speed closer to that of the chemical indicators. The present inventionprovides methods and devices for indicating sterilization efficacywhich, in most cases, can indicate sterilization failure within eighthours.

The method of the present invention comprises

a) subjecting to a sterilization cycle a source of active enzyme, saidenzyme having activity which correlates with the viability of at leastone microorganism commonly used to monitor sterilization; and

b) incubating the enzyme source, following the completion of thesterilization cycle, with an effective amount of a substrate system forthat enzyme, which system is capable of reacting with any residualactive enzyme to produce a detectable enzyme-modified product.

The reaction mixture is then evaluated in, e.g., a fluorometer or acolorimeter, to determine the presence of any enzyme-modified product.The existence of detectable enzyme-modified product above backgroundwithin an established period of time (dependent upon the identity of theenzyme and the substrate, the concentration of each, and the incubationconditions) indicates a sterilization failure. The lack of detectableenzyme-modified product within the established period of time indicatesa sterilization cycle which has been lethal to the test organism and istherefor adequate.

The source of active enzyme may be the purified enzyme isolated from anorganism, or may be a microorganism, which may itself be one commonlyused to monitor sterilization, such as Bacillus stearothermophilus orBacillus subtilis. When such a microorganism is used as the enzymesource, the method of the present invention may include the step

c) incubating any of the microorganisms which remain viable, followingthe completion of the sterilization cycle, with an aqueous nutrientmedium capable, with incubation, of promoting growth of viablemicroorganisms, and a detector material capable of undergoing adetectable change in response to growth of the microorganisms, underconditions suitable to promote growth of viable microorganisms.

The present invention further provides rapid read-out sterilityindicators useful in practicing the above-described methods. One suchsterility indicator comprises:

a) an outer container having liquid impermeable and substantiallynon-gas absorptive walls, the container having at least one openingtherein, with a gas-transmissive, bacteria-impermeable means coveringthe opening; and

b) contained within the outer container, a detectable amount of anisolated active enzyme whose activity correlates with the viability ofat least one microorganism commonly used to monitor sterilization.

Another rapid-read out sterility indicator comprises:

a) an outer container having liquid impermeable and substantiallynon-gas absorptive walls, the container having at least one openingtherein, with a gas-transmissive, bacteria-impermeable means coveringthe opening;

b) contained within the outer container, a source of active enzyme in adetectable concentration, the enzyme having activity which correlateswith the viability of at least one microorganism commonly used tomonitor sterilization; and

c) also contained within the outer container an effective amount of anenzyme substrate system capable of reacting with active enzyme toproduce a detectable enzyme-modified product.

The ability of the present invention to rapidly determine the efficacyof a sterilization cycle is based upon the discovery that

1) certain enzymes remain active following a sterilization cycle whichis marginally sufficient to kill the test microorganism whose viabilitycorrelates with the enzyme's activity; and

2) the enzyme activity following the marginal sterilization cycle issufficient to convert a substrate system for that enzyme to a detectableconcentration of product within a relatively short period of time, e.g.,generally less than about eight hours.

Where a test microorganism is used along with, or as the source of, theenzyme, very low numbers of the test microorganism can survive themarginal sterilization cycle. However, there is sufficient enzymeactivity associated with the inactivated microorganisms to indicate asterilization failure.

The enzyme detection method of the present invention acts as a fail safein marginal sterilization cycles because the enzymes of the presentinvention are more resistant to sterilization conditions than the testmicroorganism. In less complete sterilization cycles the existence ofdetectable enzyme-modified product, and, hence, the existence of enzymeactivity can be used to predict the survival or viability of the testmicroorganism if it were subjected to the same sterilization conditionsand incubated with nutrient medium for at least twenty-four hours.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a cross-sectional view of one embodiment of a sterilityindicator of the present invention, with the closure device 26 removed.

FIG. 2 is an exploded perspective view of the sterility indicator ofFIG. 1, closure device 26 included.

FIG. 3 is a cross-sectional view of a preferred embodiment of anindicator of the present invention, with closure 56 in the closedposition.

FIG. 4 is an exploded perspective view of the indicator of FIG. 3.

FIG. 5 is a cross-sectional view of another preferred embodiment of asterility indicator of the present invention, with closure 86 in theopen position.

FIG. 6 is an exploded perspective view of the sterility indicator ofFIG. 5.

FIG. 7 is a graphic representation of relative fluorescence versusincubation time for sterility indicators of this invention, afterexposure to a sterilization cycle for varying periods of time.

FIG. 8 is also a graphic representation of relative fluorescence versusincubation time for sterility indicators of this invention, afterexposure to a sterilization cycle for varying periods of time.

DETAILED DESCRIPTION OF THE INVENTION

The enzymes useful in the practice of the present invention are enzymesincluding extracellular and intracellular enzymes, whose activitycorrelates with the viability of at least one microorganism commonlyused to monitor sterilization efficacy, hereinafter referred to as a“test” microorganism. By “correlates” it is meant that the enzymeactivity, over background, can be used to predict future growth of thetest microorganism. The enzyme must be one which following asterilization cycle which is sublethal to the test microorganism,remains sufficiently active to react with a substrate system for theenzyme, within twenty-four hours, and in preferred embodiments withineight hours or less, yet be inactivated or appreciably reduced inactivity following a sterilization cycle which would be lethal to thetest microorganism.

The following test has proven useful in identifying those enzymes havingthe requisite characteristics to be useful in the sterilizationmonitoring devices and methods of the present invention. The enzyme whensubjected to sterilization conditions which would be just sufficient todecrease the population of 1×10⁶ test microorganisms by about 6 logs(i.e., to a population of about zero as measured by lack of outgrowth ofthe test microorganisms), has residual enzyme activity which is equal to“background” as measured by reaction with a substrate system for theenzyme; however, the enzyme upon being subjected to sterilizationconditions sufficient only to decrease the population of 1×10⁶ testmicroorganisms by at least 1 log, but less than 6 logs, has enzymeactivity greater than “background” as measured by reaction with theenzyme substrate system. The enzyme substrate system is a substance, ormixture of substances, which is acted upon by the enzyme to produce adetectable, e.g., fluorescent or colored, enzyme-modified product. Theenzyme activity is measured by the amount of detectable enzyme-modifiedproduct produced. Preferably, the enzyme is one which has sufficientactivity, following sterilization conditions insufficient to decreasethe population of the test microorganism by 6 logs, to react with theenzyme substrate system and produce a detectable amount ofenzyme-modified product within twenty-four hours, preferably withineight or less hours, and most preferably within two or less hours.

Preferably, the activity of the enzyme after sterilization conditionsinsufficient to decrease the microorganism population by 6 logs, is atleast 2 percent greater, and more preferably at least 5 percent greater,than background, and most preferably is at least 10 percent abovebackground. It is understood that the residual enzyme activity levelwhich is defined as “background” for purposes of this invention, may behigher than that achieved by the spontaneous conversion of enzymesubstrate to product after the enzyme has been totally and irreversiblyinactivated.

Enzymes which have been found to meet the above-described test includehydrolytic enzymes from spore-forming microorganisms. Such enzymesinclude beta-D-glucosidase, alpha-D-glucosidase, alkaline phosphatase,acid phosphatase, butyrate esterase, caprylate esterase lipase,myristate lipase, leucine aminopeptidase, valine aminopeptidase,chymotrypsin, phosphohydrolase, alpha-D-galactosidase,beta-D-galactosidase, alpha-L-arabinofuranosidase,N-acetyl-β-glucosaminidase, beta-D-cellobiosidase, alanineaminopeptidase, proline aminopeptidase, tyrosine aminopeptidase,phenylalanine aminopeptidase, beta-D-glucuronidase, and a fatty acidesterase, derived from spore-forming microorganisms, such as Candida,Bacillus and Clostridium species of microorganisms.

Particularly useful enzymes from Bacillus stearothermophilus includealpha-D-glucosidase, beta-D-glucosidase, alkaline phosphatase, acidphosphatase, butyrate esterase, caprylate esterase lipase, leucineaminopeptidase, chymotrypsin, phosphophydrolase, alpha-D-galactosidase,beta-D-galactosidase, alanine aminopeptidase, tyrosine aminopeptidase,and phenylalanine aminopeptidase and a fatty acid esterase. Particularlyuseful enzymes from Bacillus subtilis includealpha-L-arabinofuranosidase, beta-D-glucosidase,N-acetyl-β-glucosaminidase, beta-D-cellobiosidase, alanineaminopeptidase, proline aminopeptidase, tyrosine aminopeptidase, leucineaminopeptidase and phenylalanine aminopeptidase.

Beta-D-glucosidase and alpha-L-arabinofuranosidase from Bacillussubtilis are particularly useful in the monitoring of ethylene oxidesterilization. Alpha-D-glucosidase from Bacillus stearothermophilus isparticularly useful to monitor steam sterilization conditions.

The source of active enzyme may be:

1) the purified, isolated enzyme derived from an appropriatemicroorganism;

2) a microorganism to which the enzyme is indigenous or added by geneticengineering; or

3) a microorganism to which the enzyme has been added during sporulationor growth, such that the enzyme is incorporated or associated with themicroorganism, e.g., an enzyme added to a spore during sporulation whichbecomes incorporated within the spore. Preferred microorganisms whichmay be utilized as the source of an enzyme useful in the practice of thepresent invention are bacteria or fungi in either the spore orvegetative state. Particularly preferred sources of enzyme includeBacillus, Clostridium, Neurospora, and Candida species ofmicroorganisms.

When a microorganism is used as the source of active enzyme, the methodof the present invention may include the step of incubating any of themicroorganisms which remain viable, following the completion of thesterilization cycle, with an aqueous nutrient medium. Inclusion of thisstep confirms by conventional techniques whether the sterilizationconditions had been sufficient to kill all of the microorganisms in theindicator, indicating that the sterilization conditions had beensufficient to sterilize all of the items in the sterilizer. If growth ofthe microorganism is used in a conventional manner to confirm theresults of the enzyme test, the microorganism should be one which isconventionally used to monitor sterilization conditions. Theseconventionally used microorganisms are generally many times moreresistant to the sterilization process being employed than mostorganisms encountered in natural contamination. The bacterial spore isrecognized as the most resistant form of microbial life. It is the lifeform of choice in all tests for determining the sterilizing efficacy ofdevices, chemicals and processes. Spores from Bacillus and Clostridiaspecies are the most commonly used to monitor sterilization processesutilizing saturated steam, dry heat, gamma irradiation and ethyleneoxide.

Particularly preferred microorganisms commonly used to monitorsterilization conditions include Bacillus stearothermophilus andBacillus subtilis. Bacillus stearothermophilus is particularly useful tomonitor sterilization under steam sterilization conditions. The enzymealpha-D-glucosidase has been identified in spores of Bacillusstearothermophilus, such as those commercially available as “ATCC 8005”and “ATCC 7953” from American Type Culture Collection, Rockville, Md.Bacillus subtilis is particularly useful to monitor conditions of gasand dry heat sterilization. The enzyme beta-D-glucosidase has been foundin B. subtilis (e.g., commercially available as “ATCC 9372” fromAmerican Type Culture Collection).

Alternatively, in the event that isolated enzyme is utilized, or themicroorganism used as the source of the enzyme is not more resistant tothe sterilization conditions than the natural contaminants, anothermicroorganism commonly used to monitor sterilization conditions can beexposed to the sterilization cycle along with the enzyme source. Again,in such a case, the method of the present invention may include the stepof incubating any viable microorganism remaining after the sterilizationcycle with an aqueous nutrient medium to confirm the sterilizationefficacy.

The present invention, although herein described primarily in terms of asingle enzyme and/or microorganism species, should be understood torefer as well to the use of a plurality of enzymes and/or microorganismspecies. For example, a single sterility indicator may contain threetypes of isolated enzymes (which may be derived from three types ofmicroorganisms), one enzyme being resistant to heat, a second beingresistant to gaseous sterilizing media, and a third being resistant toradiation, e.g., gamma and beta irradiation. Similarly, a singlesterility indicator may contain three species of microorganisms, onespecies being resistant to heat, a second species being resistant togaseous sterilizing media, and the third species being resistant toradiation.

In the context of this application, an enzyme substrate system is bydefinition a substance or mixture of substances acted upon by an enzymeand converted into an enzyme-modified product. In general, theenzyme-modified product is a luminescent, fluorescent, colored orradioactive material. However, the enzyme substrate system can consistof a compound which when reacted with the enzyme, will yield a productwhich will react with an additional compound or composition to yield aluminescent, fluorescent, colored or radioactive material. Preferably,where the substrate system is to be included in the indicator deviceduring sterilization, the substrate must not spontaneously break down orconvert to a detectable product during sterilization or incubation. Forexample, in devices used to monitor steam and dry heat sterilization,the substrate must be stable at temperatures between about 20 and 180C.Preferably also, where the enzyme substrate system is to be includedwith conventional growth media, it must be stable in the growth media,e.g., not auto fluoresce in the growth media.

The prior art includes a number of fluorogenic and chromogenicsubstrates for the detection of enzymes of diverse origin which areknown, commercially available, and have been used in a variety ofenzymatic procedures. (M. Roth, Methods of Biochemical Analysis, Vol.17, D. Glick, Ed., Interscience Publishers, New York, 1969, p. 189; S.Udenfriend, Fluorescence Assay in Biology and Medicine, Academic Press,New York, 1962, p. 312; and D. J. R. Laurence, “Fluorescence Techniquesfor the Enzymologist”, Methods in Enzymology. Vol. 4, S. P. Colowick andN. O. Kaplan, Eds., Academic Press, New York, 1957, p. 174, incorporatedherein by reference.) There are two basic types of enzyme substratesystems described for the detection of specific enzymes. The first typeof substrate system can be either fluorogenic or chromogenic, and can begiven a chemical formula such as, AB. When acted upon by the enzyme, AB,breaks down to A+B. B, for example, would be either fluorescent orcolored. A specific example of a fluorogenic substrate of this typewould be 4-methylumbelliferyl phosphate. In the presence of the enzymephosphatase, the substrate will be broken down into4-methylumbelliferone and phosphate. Other fluorogenic substrates ofthis type include the derivatives of 4-methylumbelliferyl,7-amido-4-methylcoumarin, indoxyl and fluorescein, listed below. Anexample of a chromogenic substrate of this type is5-bromo-4-chloro-3-indolyl phosphate. In the presence of phosphatase,the substrate will be broken down into indigo blue and phosphate. Otherchromogenic substrates of this type include derivatives of5-bromo-4-chloro-3-indolyl, nitrophenol and phenolphtalein, listedbelow.

The second type of substrate system commonly used for the detection ofenzymes can be given the chemical formula CD, for example, which will beconverted by a specific enzyme to C+D. However, neither C nor D will befluorescent or colored, but D is capable of being further reacted withcompound Z to give a fluorescent or colored compound, thus indicatingenzyme activity. A specific fluorogenic example of this type is theamino acid lysine. In the presence of the enzyme lysine decarboxylase,lysine loses a molecule of CO₂. The remaining part of the lysine is thencalled cadaverine, which is strongly basic. A basic indicator such as4-methylumbelliferone can be incorporated and will fluoresce in thepresence of a strong base. A chromogenic substrate of this type would be2-naphthyl phosphate. The enzyme phosphatase, reacts with the substrateto yield β-naphthol. The liberated β-naphthol reacts with a chromogenicreagent containing 1-diazo-4-benzoylamino-2, 5-diethoxybenzene,commercially available as “Fast Blue BB Salt” from Sigma Chemical, toproduce a violet color. Other examples of this type are listed under thenaphthyl derivatives below.

Thus, from the foregoing one can readily appreciate that it is possibleto determine the presence of specific enzymes in microorganisms througha variety of approaches.

A preferred enzyme substrate system is a fluorogenic one, defined hereinas a compound capable of being enzymatically modified, e.g., byhydrolysis, to give a derivative fluorophor which has an appreciablymodified or increased fluorescence.

It is understood that the fluorogenic compounds are in themselves eithernon-fluorescent or meta-fluorescent (i.e., fluorescent in a distinctlydifferent way e.g., either by color or intensity, than the correspondingenzyme-modified products) and appropriate wavelengths of excitation anddetection, in a manner well known to users of fluorometricinstrumentation, are used to separate the fluorescence signal developedby the enzyme modification from any other fluorescence that may bepresent.

The prior art includes a number of fluorogenic substrates for enzymes ofdiverse origin which are known, commercially available, and have beenused in enzymological procedures. Among these are a variety offluorogenic 4-methylumbelliferyl derivatives (hydrolysable to4-methylumbelliferone); derivatives of 7-amido-4-methyl-coumarin, e.g.GB Patent No. 1,547,747 and European Patent No. 0,000,063 (Ajinomoto),both patents incorporated herein by reference; diacetylfluoresceinderivatives; and fluorescamine.

Useful 4-methylumbelliferyl derivatives include:

4-methylumbelliferyl-2-acetamido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside; 4-methylumbelliferylacetate; 4-methylumbelliferyl-N-acetyl-β-D-galactosaminide;

4-methylumbelliferyl-N-acetyl-α-D-glucosaminide;

4-methylumbelliferyl-N-acetyl-β-D-glucosaminide;

2′-(4-methylumbelliferyl)-α-D-N-acetyl neuraminic acid;

4-methylumbelliferyl α-L-arabinofuranoside;

4-methylumbelliferyl α-L-arabinoside; 4-methylumbelliferyl butyrate;

4-methylumbelliferyl β-D-cellobioside; methylumbelliferyl β-D-N,N′-diacetyl chitobioside; 4-methylumbelliferyl elaidate;

4-methylumbelliferyl β-D-fucoside; 4-methylumbelliferyl α-L-fucoside;

4-methylumbelliferyl β-L-fucoside; 4-methylumbelliferyl α-D-galactoside;4-methylumbelliferyl β-D-galactoside;

4-methylumbelliferyl α-D-glucoside; 4-methylumbelliferyl β-D-glucoside;

4-methylumbelliferyl β-D-glucuronide; 4-methylumbelliferylp-guanidinobenzoate; 4-methylumbelliferyl heptanoate;

4-methylumbelliferyl α-D-mannopyranoside; 4-methylumbelliferylβ-D-mannopyranoside; 4-methylumbelliferyl oleate;

4-methylumbelliferyl palmitate; 4-methylumbelliferyl phosphate;

4-methylumbelliferyl propionate; 4-methylumbelliferyl stearate;

4-methylumbelliferyl sulfate; 4-methylumbelliferyl β-D-N, N′,N″-triacetylchitotriose; 4′-methylumbelliferyl2,3,5-tri-β-benzoyl-α-L-arabinofuranoside;

4-methylumbelliferyl-β-trimethylammonium cinnamate chloride; and

4-methylumbelliferyl β-D-xyloside.

Useful 7-amido-4-methylcoumarin derivatives include:

L-alanine-7-amido-4-methylcoumarin;

L-proline-7-amido-4-methylcoumarin;

L-tyrosine-7-amido-4-methylcoumarin;

L-leucine-7-amido-4-methylcoumarin;

L-phenylalanine-7-amido-4-methylcoumarin; and7-glutaryl-phenylalanine-7-amido-4-methylcoumarin.

Useful peptide derivatives of 7-amido-4-methyl coumarin include:N-t-BOC-IIe-Glu-Gly-Arg 7-amido-4-methylcoumarin;N-t-BOC-Leu-Ser-Thr-Arg 7-amido-4-methylcoumarin; N-CBZ-Phe-Arg7-amido-4-methylcoumarin; Pro-Phe-Arg 7-amido-4-methylcoumarin;N-t-BOC-Val-Pro-Arg 7-amido-4-methylcoumarin; and N-glutaryl-Gly-Arg7-amido-4-methylcoumarin.

Useful diacetylfluorescein derivatives include fluorescein diacetate,fluorescein di-(β-D-galacto-pyranoside), and fluorescein dilaurate.

Where the enzyme whose activity is to be detected isalpha-D-glucosidase, chymotrypsin, or fatty acid esterase, e.g., fromBacillus stearothermophilus, the fluorogenic enzyme substrate is mostpreferably 4-methylumbelliferyl-alpha-D-glucoside,7-glutarylphenylalanine-7-amido-4-methyl coumarin, or4-methylumbelliferyl heptanoate, respectively. Where the enzyme whoseactivity is to be detected is alpha-L-arabinofuranosidase, e.g., derivedfrom Bacillus subtilis, a most preferred fluorogenic enzyme substrate is4-methylumbelliferyl-alpha-L-arabinofuranoside. Where the enzyme whoseactivity is to be detected is beta-D-glucosidase, e.g., derived fromBacillus subtilis, a most preferred fluorogenic enzyme substrate is4-methylumbelliferyl-beta-D-glucoside.

Another useful enzyme substrate system is a chromogenic compound capableof being enzymatically modified to give a derivative chromophor, or aproduct which reacts with another compound to give a derivativechromophor, which chromophor has a different or more intense color. Itis understood that the chromogenic compounds are in themselves eithernon-colored or colored in a distinctly different way, e.g., either bycolor or intensity, than the corresponding enzyme-modified products.Appropriate wavelengths of excitation and detection, in manners wellknown to users of colorometric instrumentation are used to separate thecolored signal developed by the enzyme modification from any other colorthat may be present.

A number of chromogenic substrates have been used in enzymologicalprocedures. Among the useful chromogenic substrates are5-bromo-4-chloro-3-indolyl derivatives; nitrophenyl derivatives; indoxylderivatives; and phenolphtalein derivatives.

Useful 5-bromo-4-chloro-3-indolyl derivatives include5-bromo-6-chloro-3-indolyl acetate, 5-bromo-4-chloro-3-indolyl acetate,5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside,5-bromo-4-chloro-3-indolyl-1,3-diacetate,5-bromo-4-chloro-3-indolyl-β-D-fucopyranoside,5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside,5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid,5-bromo-4-chloro-3-indolyl phosphate, and 5-bromo-4-chloro-3-indolylsulfate.

Useful nitrophenyl derivatives include p-nitrophenol and o-nitrophenolderivatives. Particularly useful p-nitrophenols includediethyl-p-nitrophenyl phosphate; di-p-nitrophenyl phosphate;p-nitrophenyl-2-acetamido-2-deoxy-3-O-β-galactopyranosyl-β-glucopyranoside;p-nitrophenyl-2-acetamido-2-deoxy-β-glucopyranoside; p-nitrophenylacetate; p-nitrophenyl-N-acetyl-β-D-glucosaminide; p-nitrophenyl-β-D-N,N′-diacetylchitobioside; p-nitrophenyl-α-glucopyranoside;p-nitrophenyl-α-maltoside; p-nitrophenyl-β-maltoside;p-nitrophenyl-α-mannopyranoside; p-nitrophenyl-β-mannopyranoside;p-nitrophenyl myristate; p-nitrophenyl palmitate; p-nitrophenylphosphate; bis(p-nitrophenyl)phosphate; tris(p-nitrophenyl)phosphate;p-nitrophenyl-β-glucopyranoside; p-nitrophenyl-β-glucuronide;α-p-nitrophenylglycerine; p-nitrophenyl-α-rhamnopyranoside;p-nitrophenyl stearate; p-nitrophenyl sulfate;p-nitrophenyl-2,3,4,6-tetra-O-acetyl-β-glucosaminide; p-nitrophenylthymidine mono-phosphate; p-nitrophenyl-2,3,4-tri-O-acetyl-β-glucuronicacid methyl ester; and p-nitrophenyl valerate.

Particularly useful o-nitrophenols include o-nitrophenyl acetate,o-nitrophenyl-β-glucoside and o-nitrophenyl-β-D-glucopyranoside. Otherparticularly useful nitrophenyl derivatives includenitrophenyl-β-fucopyranoside; nitrophenyl-α-galactopyranoside;nitrophenyl-β-galactopyranoside; nitrophenyl butyrate; nitrophenylcaprate; nitrophenyl caproate; nitrophenyl caprylate; nitrophenyllaurate; and nitrophenyl propionate.

Useful indoxyl derivatives include indoxyl-acetate; indoxylβ-D-glucoside; 3-indoxyl sulfate; 3-indoxyl phosphate.

Useful phenolphtalein derivatives include: phenolphthalein dibutyrate;phenolphthalein diphosphate; phenolphthalein disulfate; phenolphthaleinglucuronic acid; phenolphthalein mono-β-glucosiduronic acid;phenolphthalein mono-β-glucuronic acid; and phenolphthaleinmono-phosphate.

All of the above-described chromogenic substrates will react directlywith an appropriate enzyme to produce a chromophor.

Additional enzyme substrates containing 1-naphthyl, 2-naphthyl andNapthyl-AS-BI derivatives are usefully employed if the derivative enzymemodified product is further reacted with a chromogenic reagent, such asdiazotized dyes, e.g., 1-diazo-4-benzoylamino-2, 5-diethoxybenzene,(commercially available as “Fast Blue BB Salt” from Sigma Chemical),1-diazo-4-benzoylamino-2, 5-diethoxybenzene,p-diazo-2,5-diethoxy-N-benzoyalanine, 4-chloro-2-methylbenzene diazoniumchloride, and o-aminoazotoluene diazonium salt, to produce a chromophor.

Particularly useful 1-napthyl derivatives include1-naphthyl-N-acetyl-β-D-glucosaminide.

Particularly useful 2-naphthyl derivatives include 2-naphthyl-phosphate;2-naphthyl-butyrate; 2-naphthyl-caprylate; 2-naphthyl-myristate;L-leucyl-2-naphthylamide; L-valyl-2-naphthylamide;L-cystyl-2-naphthylamide; N-benzoyl-DL-arginine-2-naphthylamide;N-glutaryl-phenylalanine 2-naphthyl-amine; 2-naphthyl-phosphate;6-Br-2-naphthyl-α-D-galacto-pyranoside;2-naphthyl-βD-galacto-pyranoside; 2-naphthyl-2-D-glucopyranoside;6-bromo-2-naphthol-β-D-glucopyranoside;6-bromo-2-naphthyl-2-D-mannopyranoside; and2-naphthyl-α-L-fucopyranoside.

Particularly useful naphthyl-AS-BI derivatives includenaphthyl-AS-BI-phosphate; and naphthyl-AS-BI-β-D-glucuronide.

Where the enzyme whose activity is to be detected isalpha-D-glucosidase, e.g., from Bacillus stearothermophilus, thechromogenic enzyme substrate is most preferablyp-nitrophenyl-α-glucopyranoside. Where the enzyme whose activity is tobe detected is alpha-L-arabinofuranosidase, e.g., derived from Bacillussubtilis, a most preferred chromogenic enzyme substrate isp-nitrophenyl-alpha-L-arabinofuranoside. Where the enzyme whose activityis to be detected is beta-D-glucosidase, e.g., derived from Bacillussubtilis, a most preferred chromogenic enzyme substrate isp-nitrophenyl-β-D-glucopyranoside.

In order to carry out the method of the present invention, it isessential that the operator be knowledgable concerning the enzyme whoseactivity is to be detected, and the enzyme substrates which will reactwith the enzyme so as to produce a product which can be detected eitherby its fluorescence, color, etc. (See M. Roth, Methods of BiochemicalAnalysis, Vol. 17, D. Glick, Ed., Interscience Publishers, New York,N.Y., 1969, incorporated herein by reference.) The appropriate enzymesubstrate to be utilized will depend upon the identity of the enzymewhose activity is under study. Below is a list of a number of preferredfluorogenic and colorogenic enzyme substrates and an enzyme which willreact with the substrate to produce a product having appreciablymodified or increased fluorescence or color.

Substrate Enzyme Probed 4-Methylumbelliferyl acetate Esterase4-Methylumbelliferyl butyrate Esterase 4-Methylumbelliferyl elaidateLipase 4-Methylumbelliferyl-β-D- β-D-Galactosidase galactopyranoside4-Methylumbelliferyl-α-D- α-D-Galactosidase galactopoyranoside4-Methylumbelliferyl-α-D- α-D-Glucosidase glucopyranoside4-Methylumbelliferyl-β-D- β-D-Glucosidase glucopyranoside4-Methylumbelliferyl heptanoate Esterase 4-Methylumbelliferyl oleateLipase 4-Methylumbelliferyl phosphate Acid or Alkaline phosphatase4-Methylumbelliferyl propionate Esterase4-Methylumbelliferyl-β-D-galactoside β-D-Galactosidase4-Methylumbelliferyl-β-D-glucoside β-D-Glucosidase4-Methylumbelliferyl-α-D-glucoside α-D-Glucosidase4-Methylumbelliferyl-α-L- α-L- arabinofuranoside ArabinofuranosidaseL-Leucine-7-amido-4-methylcoumarin Leucine aminopeptidase7-glutaryl-phenylalanine-7- Chymotrypsin amido-4-methylcoumarinD-Melibiose α-D-Galactosidase p-Nitrophenyl phosphate Alkaline or Acidphosphatase p-Nitrophenyl acetate Lipaseo-Nitrophenyl-β-D-galactopyranoside β-D-Galactosidasep-Nitrophenyl-α-D-galactopyranoside α-D-Galactosidaseo-Nitrophenyl-β-D-glucopyranoside β-D-Glucosidasep-Nitrophenyl-α-D-glucopyranoside α-D-Glucosidasep-Nitrophenyl-β-D-glucuronide β-D-Glucuronidasep-Nitrophenyl-α-L-arabinofuranoside α-L-Arabino- furanosidasep-Nitrophenyl laurate Esterase p-Nitrophenyl myristate Esterasep-Nitrophenyl palmitate Esterase p-Nitrophenyl phosphate diNa saltAlkaline Phosphatase Phenolphthalein dibutyrate Esterase Phenolphthaleindiphosphate Acid or Alkaline phosphatase Phenolphthalein diphosphateAcid and Alkaline pentaNa salt phosphatase Phenolphthalein-β-D-β-D-Glucuronidase glucuronide Na salt Phenolphthalein-β-D-glucuronideβ-D-Glucuronidase L-Phenylalanine ethylester HCl ChymotrypsinPhenyl-β-D-galactopyranoside β-D-Galactosidase Phenyl-β-D-glucuronideβ-D-Glucuronidase Phenyl-β-D-glucopyranoside β-D-GlucosidasePhenyl-β-D-glucuronide β-D-Glucuronidase Phenyl-α-D-glucosideα-D-Glucosidase Sodium β-glycerophosphate Acid or Alkaline phosphataseSodium 1-naphthyl phosphate Acid or Alkaline phosphatase Sodium2-naphthyl phosphate Acid or Alkaline phosphatase 2-Naphthyl-butyrateEsterase β-Naphthyl acetate Lipase 6-Br-2-naphthyl-β-D-glucosideβ-D-Glucosidase L-Leucyl-2-naphthylamide Leucine aminopeptidaseL-Valyl-2-naphthylamide Valine aminopeptidaseN-glutaryl-phenylalanine-2- Chymotrypsin naphthylamineNaphthyl-AS-BI-phosphate Phosphohydralase Indoxyl acetate LipaseN-Methylindoxyl acetate Lipase N-Methylindoxyl myristate Lipase5-Bromoindoxyl acetate Lipase 3-Indoxyl phosphate Acid or Alkalinephosphatase Indoxyl-β-D-glucoside β-D-Glucosidase 5-Br-4-Cl-3-Indolylacetate Lipase 5-Br-4-Cl-3-Indolyl phosphate Alkaline or Acidphosphatase 5-Br-4-Cl-3-Indolyl-β-D- β-D-Glucuronidase glucuronic acidDiacetylfluorescein Lipase/esterase

The enzyme and its appropriate enzyme substrate are reacted in abuffered aqueous solution. The ionic conditions of the buffered solutionshould be such that the enzyme and enzyme substrate are not effected.Preferably, an isotonic buffer, such as phosphate buffered salinesolution, tris(hydroxymethyl) aminomethane-HCl solution, or acetatebuffer is chosen. These preferred isotonic buffers are compatible withmost fluorogenic and chromogenic enzyme substrates. Anotherconsideration in choosing the buffers is its influence on the enzymeactivity. For example, phosphate buffered saline contains a highconcentration of inorganic phosphate which is a strong competitiveinhibitor of alkaline phosphatase. For this enzyme, a Tris-HCl bufferis, therefore, advised.

The concentration of enzyme substrate present in the reaction mixture isdependent upon the identity of the particular substrate and enzyme, theamount of enzyme-product that must be generated to be detectable, eithervisually or by instrument, and the amount of time that one is willing towait in order to determine whether active enzyme is present in thereaction mixture. Preferably, the amount of enzyme substrate issufficient to react with any residual active enzyme present, after thesterilization cycle, within about an eight hour period of time, suchthat at least 1×10⁻⁸ molar enzyme-modified product is produced. Wherethe enzyme substrate is a 4-methylumbelliferyl derivative, it has beenfound that its concentration in the aqueous buffered solution ispreferably between about 1×10⁻⁵ and 1×10⁻³ molar.

The aqueous solution containing the enzyme substrate also preferably isadjusted to a pH of about 5.0 to 9.5, preferably about 7.5, in order toprevent autofluorescence of some basic fluorogenic substrates.

The enzyme substrate in the aqueous buffered solution is incubated withthe enzyme source whose activity is to be detected after the enzymesource has been subjected to the sterilization cycle. Incubation iscontinued for a period of time and under conditions sufficient toliberate a detectable amount of the enzyme modified product, assumingthat any of the enzyme remains active. In general, the amount of productwhich is detectable by known methods is at least 1×10⁻⁸ molar.Preferably, the incubation conditions are sufficient to generate atleast 1×10⁻⁸ molar of enzyme-modified product, more preferably, about1×10⁻⁶ to 1×10⁻⁵ molar of enzyme-modified product. The incubation timeand temperature needed to produce a detectable amount of enzyme-modifiedproduct will depend upon the identity of the enzyme and the substrate,and the concentrations of each present in the reaction mixture. Ingeneral, the incubation time required is between about 1 minute and 12hours, and the incubation temperature is between about 20 and 70° C.Preferably, where Bacillus subtilis or Bacillus stearothermophilus isthe source of the enzyme, the incubation time required is between about10 minutes and 3 hours, and the incubation temperature required isbetween about 30 and 40° C., and between about 52 and 65° C.,respectively.

Generally applicable methods for detecting enzyme modified product thatmay be used in biochemical analysis include photometric, potentiometric,gravimetric, calorimetric, conductometric, and amperometric techniques.For the purpose of this invention, fluorometric and spectrophotometricmethods of measuring detectable enzyme modified product are preferred.For example, the specific enzyme substrate may comprise a4-methylumbelliferyl derivative which on interaction with the enzymegives rise to umbelliferone which is monitored fluorimetrically, or thesubstrate may comprise a nitrophenol, or similar type of derivative,which on interaction with the enzyme gives rise to a colored productwhich is monitored calorimetrically.

The procedure of the invention permits very rapid detection of enzymeactivity, which can be used to predict conditions permitting survival ofmicroorganisms and, thus, sterilization efficacy. The enzymedetermination tests used generally require only a relatively shortperiod of incubation, e.g., from about ten minutes up to about threehours, usually from about 30 to about 90 minutes to provide sufficientenzyme modified product for detection, e.g., by spectroscopicmeasurements.

In its simplest form, a sterility indicator useful in practicing themethod of the present invention includes a source of active enzyme in acontainer having liquid impermeable and substantially gas non-absorptivewalls. The container has at least one opening, to permit sterilizingmedia to come in contact with the enzyme and a gas-transmissive,bacteria-impermeable means covering the opening. Optionally, theindicator may include within the container:

1) a microorganism commonly used to monitor sterilization efficacy,either as the source of the enzyme or in addition to the source of theenzyme;

2) a carrier upon which is coated the source of active enzyme, alone orin combination with a sterilization resistant microorganism;

3) a substrate for the active enzyme and an aqueous reaction medium forthe enzyme and it substrate; and

4) In the event that a sterilization resistant microorganism isutilized, nutrient growth media and a growth indicator. Sterilityindicators similar to those described in U.S. Pat. Nos. 3,346,464;3,585,112; 3,846,242; 4,291,122; 4,461,837; 4,416,984; 4,596,773;3,440,144; 4,528,268; 2,854,384; 3,239,429; 3,752,743; 4,304,869;4,579,823; and 4,580,682 may be usefully employed if an enzyme useful inthe practice of the present invention is included in place of, or inaddition to, the sterility resistant microorganism.

The following description is directed to Applicants' preferredembodiments. Many variations of the following devices are possible whichwill nonetheless fall within the scope of the present invention.

Referring now to FIGS. 1 and 2, a preferred sterility indicator is shownhaving an outer container in the shape of cylindrical tube 10, havingsubstantially gas non-absorptive and liquid impermeable walls 12 and anopen end 14. Tube 10 contains an enzyme carrier 16, such as a strip offilter paper, bearing a predetermined amount of active isolated enzymeand/or a predetermined number of viable microorganisms. Tube 10 alsoincludes a normally sealed, pressure-openable inner container 18, suchas a frangible glass ampoule, containing a suitable enzyme substratedissolved or suspended in an aqueous buffered solution 20, andoptionally including an aqueous nutrient growth medium. Preferably, theenzyme substrate is stable at temperatures between about 20 and 180° C.and is capable of reacting with active enzyme to yield a luminescent,fluorescent, colored or radioactive material. The aqueous nutrientmedium is capable, with incubation, of promoting growth of viablemicroorganisms when contacted therewith. The inner container 18 ispreferably snuggly retained within the outer container 10 so that verylittle of the volume of the outer container remains unoccupied. Theglass ampoule 18 is separated from the wall 12 of the tube 10 by thefilter paper carrier 16. The open end 14 of the tube 10 is provided witha gas-transmissive, bacteria-impermeable closure member 22, such as asheet. The sheet 22 may be sealed to the open end 14 of the tube 10 by,e.g., heat or adhesive sealing, or by means of a closure device 26, suchas a cap, (shown removed in FIG. 1) which has an aperture 28therethrough. During sterilization with a gaseous sterilization agent,the gaseous sterilant permeates the sheet 22 and passes through theinterior of the outer container to contact the enzyme carrier 16.

As shown in FIG. 2, the apparatus of FIG. 1 may be easily assembled bysequentially inserting into the open end 14 of the tube 10 the activeenzyme carrier 16 and the flangible glass ampoule 18, and sealing theopen end 14 of the tube with the sheet 22 by placing sheet 22 over openend 14 and then placing cap 26 over sheet 22, in closing engagement withtube 10.

Outer container 10 is made from material which will withstand the hightemperatures encountered in steam sterilizers. Conventional steamsterilizers generally reach temperatures on the order of 121° C.-135° C.Additionally, the walls of container 10 must be substantiallyimpermeable to gases and liquids. Outer container 10 which contains thecarrier 16 which is coated with viable microorganisms or active isolatedenzyme, and in which the residual active enzyme reacts with the enzymesubstrate contained in pressure-openable inner container 18, ispreferably translucent (including “transparent”) so that a change influorescence or color may be visually observed without disassembling theindicator device. Preferably, also, the outer container 10 issufficiently deformable so that the pressure-openable inner compartment18 is ruptured when the outer compartment 10 is deformed, by usingexternal pressure. Container 10 can be made by injection molding orextruding suitable materials, including polycarbonate, polypropylene,polyamides, polymethylpentenes and various polyesters. Polypropylene isthe preferred material. These materials are sufficiently temperatureresistant to withstand steam or dry heat sterilization cycles,non-absorbent of gaseous sterilizing media, liquid-impermeable,translucent or transparent and deformable.

The closure device 26 can be made from any material that will withstandthe sterilization temperatures. As in the case of the container 10,suitable materials include polycarbonate, polypropylene, polyamides,polymethylpentenes and various polyesters, with polypropylene beingpreferred.

The active isolated enzymes and/or the microorganisms which are employedin the present invention normally are carried on a suitable carrier 16.It is contemplated, however, that the enzyme and/or microorganism may becarried by the inner walls of the outer container 10, or the outer wallsof the inner container 18. Preferably, however, the isolated enzymeand/or microorganism are carried by the same or separate enzymecarriers. The enzyme carrier preferably is water-absorbent, such asfilter paper, and should not inhibit microorganism growth or enzymeactivity. Sheet-like materials such as cloth, nonwoven polypropylene,rayon or nylon, and microporous polymeric materials are especiallypreferred. However, metal foil substrates, for example, aluminum orstainless steel may be used, as well as substrates of glass (e.g., glassbeads or glass fibers), porcelain, or plastic. Additionally, the enzymecarrier can be constructed of a combination of materials such as papersecured to a plastic or glass backing strip.

To assure reproducibility, it is desired that outer container 10 containa predetermined amount of active enzyme. This is readily accomplishedwith isolated enzyme by using general methods of protein purification,such as salt fractionation, chromatography and electrophoresis asdescribed in Colowick, S., and Kaplan, N. O. (Eds), Methods inEnzymology, Academic Press, New York, Vols. I-VII, (1957-1964),incorporated herein by reference. Preferably the initial concentrationof isolated enzyme is between about 1×10⁻¹⁰ and 5×10⁻² units, morepreferably between about 1×10⁻⁸ and 5×10⁻³ units of enzyme, and mostpreferably between about 1×10⁻⁷ and 1×10⁻³ units. Where a microorganismis utilized, it is likewise desirable to use a predetermined approximatenumber of microorganisms. This is accomplished with bacterial or fungalspores by preparing a spore suspension having a known volumetric sporeconcentration, moistening the carrier 16 (e.g., filter paper) with asmall, predetermined volume of the suspension, and drying the carrier.This method permits the approximate number of spores contained on thecarrier to be easily calculated. Other methods, of course, may also beemployed. Where the microorganism is utilized as the source of theenzyme, the microorganism population which should be used will depend onthe activity of the enzyme in that organism. The enzyme activity isdependent upon the culture conditions and strain selection of themicroorganism, but can be regulated by adjusting the microorganismpopulation. Where the microorganism is Bacillus stearothermophilus orBacillus subtilis the number of microorganisms necessary to producesufficient enzyme is provided by about 1×10⁸ to 1×10² microorganisms.Where the source of enzyme is B. stearothermophilus, about 1×10³ to1×10⁷ microorganisms is preferred. Where B. subtilis is the source ofthe enzyme, about 1×10⁶ to 1×10⁸ microorganisms is preferred.

When a microorganism is used as the source of active enzyme, theincubation of the device can be continued after the time required toproduce detectable enzyme-modified product, in order to confirm byconventional techniques whether the sterilization conditions had beensufficient to kill all of the microorganisms in the indicator,indicating that the sterilization conditions had been sufficient tosterilize all of the items in the sterilizer. If growth of themicroorganism is used in a conventional manner to confirm the results ofthe enzyme test, the microorganism should be one which is conventionallyused to monitor sterilization conditions. These conventionally usedmicroorganisms are generally many times more resistant to thesterilization process being employed than most organisms encountered innatural contamination. Preferred microorganisms include Bacillisstearothermophilus and Bacillus subtilis.

Alternatively, in the event that isolated enzyme is utilized, or themicroorganism used as the source of active enzyme is not more resistantto the sterilization conditions than the natural contaminants, anothermicroorganism commonly used to monitor sterilization conditions can beincluded within container 10. In such a device, the isolated enzyme, orthe microorganism from which a useful enzyme may be derived, is used toobtain a reading of enzyme activity within generally about 10 minutes to3 hours after incubation, and the commonly used microorganism is furtherincubated in nutrient media for at least about 24 hours to confirm theenzyme activity results.

An aqueous solution of the appropriate enzyme substrate is normallyincluded in pressure-openable inner container 18. However, it iscontemplated that the enzyme substrate in dry form could be included inouter container 10 along with enzyme carrier 16. In fact, the activeenzyme and its substrate could be present in dry form in the samecarrier 16. In this construction, inner container 18 would preferablycarry the aqueous reaction medium necessary for the active enzyme andits substrate to react.

Preferably, when a microorganism commonly used to monitor sterilizationis included in the device (either as the source of the active enzyme, orin addition to the source of active enzyme) and confirmation ofmicroorganism survival by conventional methods is desired, innercontainer 18 contains an aqueous solution of the enzyme substrate andnutrient growth media. Preferably, the nutrient growth media iscompatible with most fluorogenic and chromogenic enzyme substrates andis not a competitive inhibitor for the enzyme. The types of nutrientmedia usefully employed in the present invention are widely known to theart. Examples of preferred nutrient media are aqueous solutions ofsoybean-casein digest broth, fluid thioglycollate and Dextrose Tryptone(Difco Laboratories, Inc.). A modified tryptic soy broth base, withoutglucose, is especially preferred. To avoid contamination, such aqueousnutrient media normally is sterilized after having been placed in theinner compartment. Commonly known microbial growth indicators, whichchange color in the presence of viable microorganisms, are preferablypresent in at least one of the containers. The growth indicator materialpreferably is soluble in, and imparts color (upon microorganism growth)to, the aqueous nutrient medium so that a change in color may be easilyobserved through the translucent walls of the outer container. Inaddition, the growth indicator material is preferably selected so thatit will not interfere with the color or luminescence of theenzyme-modified product. Growth indicator materials which may beemployed in the present invention are well known to the art and includepH-sensitive dye indicators (such as bromthymol blue, brom cresolpurple, phenol red, etc.), oxidation-reduction dye indicators (such asmethylene blue, etc.). Such materials commonly undergo changes in colorin response to a phenomenon of microorganism growth, such as changes inpH, oxidation-reduction potentials, etc.

The inner container 18 which contains the aqueous solution of enzymesubstrate and/or which contains the aqueous nutrient medium, is ofmaterial which is impermeable to gases and liquids and is capable ofbeing opened upon the application of pressure thereto (i.e., “pressureopenable”) to permit the enzyme substrate and/or nutrient medium toenter the outer container. The inner container is preferably offrangible material, such as glass, and, as mentioned above, ispreferably snugly carried within the outer container in coactingrelationship therewith to permit breakage or crushing of the innercontainer when the outer container is deformed. In another embodiment,the inner container may be sealed with a plug such that the plug isexpelled to release the contents of the inner container upon applicationof pressure. In still another embodiment, the closure 26 may include anampoule crushing device, as shown in U.S. Pat. No. 4,304,869, whereinthe closure has tabs depending from the bottom of the closure devicewhich upon depression of the closure device serve to crush the ampoule.Similarly, the device of the present invention may be used in a systemhaving an ampoule crushing pin disposed in the bottom of the outercontainer 10.

The active enzyme-containing outer container 10 has at least one openingtherein to permit the sterilant (e.g., steam, ethylene oxide) to contactthe source of active enzyme during sterilization. This opening isnormally closed or plugged with a gas-transmissive, bacteria-impermeablemeans. Suitable means include closure member 22, made of fibrousmaterials such as cotton, glass wool, cloth, nonwoven webs made frompolypropylene, rayon, polypropylene/rayon, nylon, glass or other fibers,filter papers, microporous hydrophobic and hydrophilic films, opencelled polymeric foams, and semi-permeable plastic films such as thosedescribed in U.S. Pat. No. 3,346,464. Fibrous or cellular materials arepreferred because of the ease with which such materials transmitsterilizing gases. Preferred closure member materials includehydrophobic materials such as nylon web, microporous hydrophobic film,or glass fiber nonwoven web. Especially preferred is a microporoushydrophobic film, commercially available from Celanese SeparationsProducts, Charlotte, North Carolina, under the trade name “Celgard®K-442 Microporous Film”. In effect, the fibrous or cellular closuremembers serve as filters for bacteria and fungi and hence should havepore sizes no larger than about 0.5 microns (e.g., be capable ofpreventing the passage therethrough of particles having dimensionslarger than about 0.5 microns). Alternatively, the closure means may bea tortuous pathway that is bacteria-impermeable, such as that describedin U.S. Pat. No. 4,461,837, incorporated herein by reference, and incommonly assigned copending U.S. Pat. No. 4,883,641, issued Nov. 28,1989.

A preferred embodiment of a sterilization indicator of the presentinvention is illustrated in FIGS. 3 and 4. The device includes an outercontainer 40, having substantially gas non-absorptive and liquidimpermeable walls 42 and an open end 44. The outer container 40 includesa pressure-openable inner container 48 which contains an aqueoussolution 50 of a suitable enzyme substrate, preferably in admixture withan aqueous nutrient medium. The open end 44 of the outer container 40 iscovered by a gas-transmissive, bacteria-impermeable closure member 52.With that, the similarity between the device depicted in FIG. 1 ends. Inthe device of FIGS. 3 and 4, the enzyme carrier 46 is located at thebottom closed end of the outer container 40, and a barrier 47 ispositioned like a plug between the enzyme carrier 46 and thepressure-openable inner container 48. The barrier 47 is preferably madeof materials which are non-fluorescent, for use with fluorogenic enzymesubstrates, such as nonwoven webs made from fibers such as cotton,rayon, polypropylene, polypropylene/rayon blends, nylon or glass. Mostpreferably barrier 47 is constructed from a polypropylene nonwoven web,such as “Thinsulate® 200-B brand Thermal Insulation”, commerciallyavailable from 3M, St. Paul, Minn.

Barrier 47 serves to isolate the enzyme carrier 46 from the innercontainer 48, thus eliminating cold spots where the ampoule 48 may bepositioned over the carrier 46. The existence of cold spots can causecondensation to collect on the enzyme carrier. The condensate may effectthe activity of the enzyme contained on the carrier 46. Barrier 47 ispreferably made from a hydrophobic material so that enzyme-modifiedproduct concentrates around the enzyme carrier and does not diffuserapidly into the area of the container which is on the other side of thebarrier. Maintaining a higher concentration of the enzyme-modifiedproduct in the lower portion of the indicator enables the enzymemodified product, whether it be luminescent or colored to be detectedafter a shorter period of incubation than would be the case if thecarrier 46 was reacted with the entire contents of inner container 48.In general, as illustrated in Example 1, preferred devices whichincorporate a barrier 47, provide reliable information on sterilizationefficacy within about 10 minutes. Similar devices, not utilizing such abarrier, require about two hours to provide reliable sterilizationefficacy information.

The closure 56 is comprised of a top 57 and depending sidewalls 59. Theclosure has a hollow body open at the bottom, with the interior diameterof the closure being about equal to the exterior diameter of outercontainer 40, so that closure 56 may be frictionally engaged over theopen end 44 of outer container 40. Cut within the sidewalls 59 arepreferably a plurality of windows 58. When the indicator device isplaced in a load to be sterilized, the closure 56 is placed over theopening in the outer container in such a manner that the exteriorsidewalls 42 of the outer container do not block windows 58. In such aposition, sterilant in the sterilizer may enter container 40 by flowingthrough windows 58. Upon completion of the sterilization cycle, theclosure may be fully inserted by depressing it to force the sidewalls 42of the outer container into engagement with the interior surface of top57 thereby blocking windows 58. The interior of the container 40 is thensealed from the outside environment.

FIGS. 5 and 6 illustrate an alternative preferred embodiment of thesterilization indicator of the present invention. The device includes,as does the device of FIGS. 3 and 4, an outer container 70, with gasnon-absorptive and liquid impermeable walls 72 and an open end 74; apressure-openable inner container 78 within outer container 70containing an aqueous solution of enzyme substrate 80, preferably inadmixture with nutrient growth medium; and gas-transmissive,bacteria-impermeable closure member 82, which is held over the open end74 of the outer container by cap 86. The enzyme carrier 77 within outercontainer 70 is attached to a wick strip 76 by, for example, adhesive orheat sealing. The wick strip can be made from any water-absorbentmaterial, such as filter paper, cloth, or rayon. Additionally, the wickstrip can be constructed of a combination of materials such as papersecured to a plastic or glass backing strip. Preferably wick strip 76 isprepared from polyethylene coated paper. Preferably, the dimensions ofthe wick strip and the placement of the enzyme carrier on the wick stripare such that when the inner-container is ruptured the liquid therein iscontained within the lower portion of the outer container and below theenzyme carrier 77. The aqueous enzyme substrate solution 80 travels upthe wick strip 76 to enzyme carrier 77. The enzyme-modified productconcentrates on the enzyme carrier 77 and its presence is detected in ashorter period of time than would be the case if the carrier was exposedto the entire solution present in the inner container 78.

In use, the sterility indicator depicted in FIGS. 3 and 4 is placed in asterilizer chamber together with a number of items to be sterilized by,for example, steam or ethylene oxide gas. When the indicator is in thesterilizer, the closure 56 is in the open position, such that windows 58are open permitting entry of the sterilant. When the sterilizing agentis introduced into the chamber, the sterilant permeates through theclosure member 52 and passes barrier 47 to inactivate the enzyme andkill the test microorganisms present on carrier 46. At the end of thesterilization cycle, the sterilant is replaced with filtered air. Thesterility indicator is withdrawn from the sterilizer, the closure 56 isfully inserted to block windows 58, and glass ampoule 48 is broken by,for example, finger pressure, causing the aqueous solution of enzymesubstrate and nutrient growth media to contact the enzyme carrier 46.The indicator is then placed in a suitable incubating environment (e.g.the indicator may be incubated at about 56° C. for about 10 minutes to 2hours). Any enzyme not inactivated by the sterilant will react with itssubstrate, producing a preferably colored or fluorescent enzyme-modifiedproduct. The occurrence of a change in color or fluorescence is observedor measured spectrophotometrically through the translucent walls 42 ofthe outer container 40, and indicates that the sterilization cycle hadnot inactivated all the active enzyme on the carrier 46. The presence ofactive enzyme predicts survival and eventual outgrowth of the testmicroorganism and indicates that the sterilization cycle was perhapsinsufficient to completely sterilize the items in the sterilizer. Theabsence of any change in color or fluorescence indicates that thesterilization cycle had been sufficient to inactivate all of the enzymeon the carrier 46, and hence was sufficient to sterilize the items inthe sterilizer. With further incubation of the device (at 56° C. forabout 24 to 48 hours) the early prediction of test microorganismsurvival can be confirmed by the existence color changes in the growthmedia.

A preferred method of monitoring the fluorescence of an indicator ofthis invention is a fluorimeter designed specifically for the devicesdescribed in this invention. A fluorimeter eliminates the subjectiveinterpretation encountered when attempting to visually differentiatebetween low levels of fluorescent product and background or nofluorescence. A fluorimeter can be calibrated to detect a minimum amountof fluorescent product within a given incubation period.

A particularly preferred fluorimeter, designed for use with the devicesof this invention, consists of a chamber designed to block ambient lightwhile positioning the outer container of the indicator such that theenzyme carrier within can be illuminated with a 365 nm wavelengthultraviolet light, and a photodiode can detect any resultantfluorescence in the 460 nm wavelength region. The fluorimeter iscalibrated to detect at least 1.0×10⁻⁵ M 4-methylumbelliferone.

Several methods can be used to differentiate the fluorescent positivedevices from the non-fluorescent or negative devices. In the firstapproach, a fluorescent threshold limit equivalent to the fluorescenceproduced by 1×10⁻⁵ M 4-methylumbelliferone is established in thefluorimeter. When a test sample with sufficient active enzyme convertsenough substrate to exceed the threshold limit, after the enzyme carrieris allowed to react with the substrate at e.g., 56° C. for 15 minutes,the fluorimeter indicates a positive sample by illuminating, forexample, a red light. If the fluorescent product produced by reaction ofthe enzyme and its substrate does not exceed the threshold limit, afterthe 15 minute incubation, for example, the fluorimeter will indicate anegative or non-fluorescent sample, with, for example, a green light.

In the second approach, the fluorimeter measures the initialfluorescence, at the beginning of the incubation period. The fluorimeterchamber is heated to the optimum temperature for the specific enzymebeing tested in the device. In the case of the enzymealpha-D-glucosidase derived from Bacillus stearothermophilus, thetemperature is 56° C. During the incubation period, the fluorimetercontinues to monitor the fluorescence and will indicate a positivefluorescent sample when at least a 5% increase in intensity above theinitial fluorescence is detected, by, for example, a red light. If lessthan a 5% increase occurs within the established incubation time, thefluorimeter will indicate a negative or non-fluorescent sample by, forexample, activating a green light.

The sterility indicator of the present invention has been describedprimarily with reference to sterilizing media such as ethylene oxide,steam and the like. The indicator is not, however, limited to theseuses, and may as well be used to indicate the efficacy of othersterilizing media, such as dry heat, radiation, propylene oxide, methylbromide, ozone, chlorine dioxide, formaldehyde, and other gaseous andliquid agents.

The invention will be illustrated by the following non-limitingexamples, in which all percentages are percent by weight, unlessotherwise indicated.

EXAMPLE 1

This example illustrates the correlation between the fluorescent resultsof the enzymatic detection method of the present invention, and theresults of conventional spore survival methods of detection. Thisexample also illustrates the shorter read-out times which can beachieved with the device illustrated in FIGS. 3 and 4, which utilizes abarrier 47 between the ampoule 48 and the enzyme carrier 46.

Bacillus stearothermophilus, commercially available as “ATCC 7953” fromAmerican Type Culture Collection, Rockville, Md., was grown overnight(16 hours) at 58° C. in tryptic soy broth. This culture was used toinoculate the surface of agar plates consisting of 8 g/l nutrient broth,4 g/l yeast extract, 0.1 g/l manganese chloride and 20 g/l agar at pH7.2. Plates were incubated at 58° C. for 72 hours. Spores were scrapedfrom the plates and suspended in sterile distilled water. The sporeswere separated from the vegetative debris by centrifuging the suspensionat 7000 rpm and 4° C. for 20 minutes. The supernatant was poured off andthe spores were resuspended in sterile distilled water. This cleaningprocedure was repeated several times. The Bacillus stearothermophilusspores were coated on 6.35 mm (¼ inch) in diameter filter paper discs,commercially available as “S&S #903 Grade Filter Paper” from Schleicher& Schuell, Inc., Keene, N.H., at a population of 1.6×10⁶ spores perdisc. This was accomplished by preparing a suspension of the B.stearothermophilus spores in water at a concentration of 1×10⁸ spore/ml,pipetting 10 μl of this suspension on each filter paper disc andallowing the discs to dry.

Two types of devices were constructed as follows. The first device wasconstructed as illustrated in FIGS. 3 and 4, with the spore coated strip46 on the bottom of the outer compartment 40 and a barrier 47 betweenthe enzyme substrate-containing ampoule 48 and the spore strip. A 1.75mm ({fraction (11/16)} inch) diameter disc of polypropylene blownmicrofiber material, with a weight of 200 g/sq. meter, commerciallyavailable as “Thinsulate® 200-B brand Thermal Insulation” from 3M, St.Paul, Minn., was used as the barrier 47. The ampoule 48 contained 0.67ml nutrient medium, consisting of 17 g of a bacteriological peptone and0.17 g of L-alanine, as well as 0.1 g4-methylumbelliferyl-alpha-D-glucoside, commercially available fromSigma Chemical Company, St. Louis, Mo., dissolved in 200 μl of N,N-dimethylformamide, and 0.03 g bromocresol purple pH indicator dye, perliter of water. The pH of the enzyme substrate and nutrient mediumsolution was adjusted to 7.6 with 0.1 N sodium hydroxide.

The outer vial 40 the cap 56 are both made from polypropylene. The outervial was 5.08 cm (2.0 inches) long, with an outer diameter of 85.1 mm(0.335 inches) and an internal diameter of 77.0 mm (0.303 inches). Thecap was 1.275 cm (0.510 inch) long with an internal diameter of 83.3 mm(0.328 inch). The inner ampoule 48 was made of glass and was 3.96 cm(1.56 inches) long, with an outer diameter of 65.5 mm (0.258 inches) anda wall thickness of 2.5 mm (0.010 inches). The closure member 52 was a1.27 mm (½ inch) in diameter piece of polypropylene, commerciallyavailable as “Celgard® K-442 Microporous Film”, from CelaneseSeparations Products, Charlotte, N.C.

The second device was identical to the first device, except that thebarrier 47 was omitted.

Five unit batches of both types of devices were placed in metalinstrument trays and exposed at 132° C. in a gravity displacement steamsterilizer, commercially available as an “Amsco Eagle™ Model 2013Sterilizer”, from American Sterilizer Company, Erie, Pa., for 0.5, 1.0,1.5, 2.0,. 2.5 and 3.0 minutes. After exposure the inner ampoulescontaining the enzyme substrate and nutrient medium were crushed and theunits were incubated at 56° C. An ultraviolet light (λ=366 nm) was usedto illuminate the vials for visually read fluorescence after 10 min., 20min., 30 min., 60 min., 120 min., 180 min., and 240 minutes ofincubation. Additionally, spore growth, as indicated by a color changefrom purple to yellow, was determined visually after 24 hours ofincubation at 56° C.

The results are reported in Table I.

TABLE I FLUORESCENCE Exposure Incubation Time (Minutes) at 56? C. SporeTime Number Positive/5 Tested Growth Device (minutes) 10 20 30 60 120180 240 at 24 hr. 1 0.5 5 5 5 5 5 5 5 5 (Spore Strip 1.0 5 5 5 5 5 5 5 5on Bottom 1.5 5 5 5 5 5 5 5 5 with Barrier 2.0 3 3 3 3 3 3 3 3 Material)2.5 0 0 0 0  2*  2*  3* 0 3.0 0 0 0 0 0 0 0 0 2 0.5 0 0 0 5 5 5 5 5(Spore Strip 1.0 0 0 0 5 5 5 5 5 on Bottom 1.5 0 0 0 5 5 5 5 5 Without2.0 0 0 0 5 5 5 5 5 Barrier 2.5 0 0 0 0  3*  3*  3* 1 Material) 3.0 0 00 0 0 0 0 0 *Indicates one or more false positive reactions which appearto be the result of residual enzyme activity following sporeinactivation in marginal sterilization cycles

The data in Table I illustrates that the presence of activealpha-D-glucosidase can be detected by the methods of the presentinvention much more quickly than can the detection of viable spores. Thedata illustrates that the detection of active enzyme in a device can beused to predict eventual spore growth in the device.

The data in Table I illustrates that using device 1 (with a barrierbetween the ampoule and the spore strip) enzyme activity can be detectedafter 10 minutes for all units which show spore growth after 24 hours ofincubation. Using device 2 (without barrier material), 2 hours ofincubation is required in order to detect enzyme activity in all unitswhich show spore growth after 24 hours of incubation.

EXAMPLE 2

Devices were constructed as illustrated in FIGS. 5 and 6, with theenzyme carrier 77 on a wick 76. One spore strip, prepared in accordancewith Example 1, was heat sealed to one end of a 0.10 mm thickpolyethylene coated paper, 6.35 mm×28.58 mm. The ampoule 78 contained 5ml/l of a nonionic surfactant, commercially available as “Tween™ 80Polyoxyethylene Sorbitan Monooleate”, from ICI Americas, Inc.,Wilmington, Del., to aid in the wetting of the spore carrier, as well asthe enzyme substrate, nutrients and indicators present in the solutionof Example 1. The outer vial, cap and inner ampoule were identical tothose used in the devices of Example 1. The closure member was asterilization grade filter paper.

Five unit batches of devices were placed in metal instrument trays andexposed at 132° C. in a gravity displacement steam sterilizer,commercially available as an “Amsco Eagle Model™ 2013 Sterilizer”, fromAmerican Sterilizer Company, Erie, Pa., for 0.5, 1.0, 1.5, 2.0,. 2.5 and3.0 minutes. After exposure the inner ampoules were crushed and thedevices were incubated at 56° C. An ultraviolet light (λ=366 nm) wasused to illuminate the vials for visually read fluorescence after 10min., 20 min., 30 min., 60 min., 120 min., 180 min., and 240 minutes ofincubation. Additionally, spore growth, as indicated by a color changefrom purple to yellow, was determined visually after 24 hours ofincubation.

The results are reported in Table II.

TABLE II FLUORESCENCE Exposure Incubation Time (Minutes) at 56° C. TimeNumber Positive/5 Tested 24 hr. (minutes) 10 20 30 60 120 180 240 Growth0.5 5 5 5 5 5 5 5 5 1.0 5 5 5 5 5 5 5 5 1.5 5 5 5 5 5 5 5 5 2.0 0 0 0 0 4*  5*  5* 0 2.5 0 0 0 0 0 0 0 0 3.0 0 0 0 0 0 0 0 0 *Indicates one ormore false positive reactions which appear to be the result of residualenzyme activity following spore inactivation in marginal sterilizationcycles

The data in Table II illustrates that much quicker detection of sporesurvival can be achieved using the devices and enzyme detection methodsof the present invention in contrast to standard 24 hour spore growth.

EXAMPLE 3

This example illustrates several enzymes, associated with Bacillusstearothermophilus, which are useful in the practice of the presentinvention. In this example an enzyme substrate kit, commerciallyavailable as “API-XYM™ System” from API Analytab Products, Plainview,N.Y., was utilized. This kit consists of 19 different dehydrated,chromogenic enzymatic substrates, packed individually in a series ofmicrocupules. The addition of an aqueous sample to each microcupulerehydrates the substrate. The test kit is incubated for a desiredinterval and the reactions are visualized after the addition of thedetector reagents supplied with the system.

Devices made according to Device 1 of Example 1, were exposed for 1minute or 3 minutes at 132° C. in the steam sterilizer used inExample 1. It was determined that the Bacillus stearothermophilus sporeswhen used in this device will survive a one-minute exposure and bekilled after 3 minutes. After exposure the spore strips were asepticallytransferred to each microcupule in the enzyme substrate kit and 50 μl ofsterile distilled water was added to each well. One kit contained thespore strips exposed for 1 minute, a second kit contained the sporestrips exposed for 3 minutes and a third kit contained unexposed sporestrips.

The kit containing the unexposed strips was incubated at 56° C. for 5hours. The kits containing exposed spore strips were allowed to incubateat 56° C. for 7 hours. After incubation, the detector reagents A and B(available with the “API-XYM™ System”) were added for color developmentof any enzymatic reactions occurring in the microcupules of each kit.

The detection of color in each microcupule of each kit is reported inTable III. A number of the enzymes showed readily detectable activityafter 1 minute exposure, and no or substantially decreased activityafter 3 minutes exposure. Several of the enzymes were not indiginous toB. stearothermophilus and did not illustrate detectable activity in theunexposed state. Several other enzymes (including myristate lipase,valine aminopeptidase, chymotrypsin, and beta-glucuronidase) which didnot illustrate detectable activity in the unexposed state, apparentlywere activated by the exposure to heat. It is believed that theseenzymes which show detectable activity after a 1 minute exposure and noactivity or reduced activity after 3 minutes exposure, could be usefullyemployed in the present invention, even if no enzyme activity isdetected in the unexposed state.

TABLE III Enzyme Spore Strip Exposure Assayed For Unexposed 1 Minute 3Minute Negative control − − − Alkaline phosphatase +2 +3 − Butyrateesterase +4 +5 − Caprylate esterase lipase +2 +3 − Myristate lipase − VW− Leucine aminopeptidase +1 +5 − Valine aminopeptidase − VW − Cystineaminopeptidase − − − Trypsin − − − Chymotrypsin − +4 − Acid phosphatase+3 +4 +1 Phosphophydrolase +4 +5 +3 Alpha-galactosidase +5 +5 −Beta-galactosidase − − − Beta-glucuronidase − VW − Alpha-glucosidase +5+5 − Beta-glucosidase +4 VW − N-acetyl-beta- − − − glucosaminidaseAlpha-mannosidase − − − Alpha-fucosidase − − − − = No color developmentVW = Very weak color development +1 = Weak color development +2,3,4 =Intermediate color development (color development increasing withincreasing number) +5 = Strong color development

Table III illustrates that a number of enzymes present in B.stearothermophilus, including alkaline phosphatase, butyrate esterase,caprylate esterase lipase, myristate lipase, leucine aminopeptidase,valine aminopeptidase, chymotrypsin, acid phosphatase, phosphohydrolase,alpha-galactosidase, beta-glucuronidase, and alpha-glucosidase,B-glucosidase, have sufficient activity following a sublethalsterilization exposure to be detected before growth of the spore isdetected.

EXAMPLE 4

Bacillus stearothermophilus spores, prepared in accordance with Example1, were coated and dried on 6.35×28.58 mm (¼×⅜ inch) carriers made of afilter paper, commercially available as “S&S #591A Grade Filter Paper”from Schleicher and Schuell, Inc. of Keene, N.H. at concentrations of1.0×10⁷, 7.5×10⁵, 1.0×10⁵, 1.7×10⁴, 2.8×10³ spores per carrier. Deviceswere assembled using these spore strips, as shown in FIGS. 3 and 4 anddescribed as Device 1 in Example 1.

Three unit batches of devices were placed in metal instrument trays andexposed at 132° C. in a gravity displacement steam sterilizer,commercially available as an “Amsco Eagle™ Model 2013 Steam Sterilizer”,from American Sterilizer Company, Erie, Pa., for 1.0, 1.5, 2.0,. 2.5 and3.0 minutes. After exposure the inner ampoules were crushed and thedevices were incubated at 56° C. An ultraviolet light (λ=366 nm) wasused to illuminate the vials to visually detect fluorescence after 10min., 20 min., 30 min., 60 min., 120 min., 180 min., 240 min., 300 min.and 360 minutes of incubation. Additionally, spore growth, as indicatedby a color change from purple to yellow, was determined visually after24 hours of incubation.

The results are reported in Table IV.

TABLE IV FLUORESCENCE Exposure Incubation Time (Minutes) at 56° C. SporeSpore Time Number Positive/3 Tested Growth Population (minutes) 15 30 4560 120 180 240 300 360 at 24 hr. 1.0 × 10⁷ 1.0 3 3 3 3 3 3 3 3 3 3 1.5 33 3 3 3 3 3 3 3 3 2.0 0 0  1*  1*  1*  2*  2*  2*  2* 0 2.5 0 0 0 0 0 00 0 0 0 3.0 0 0 0 0 0 0 0 0 0 0 7.5 × 10⁵ 1.0 3 3 3 3 3 3 3 3 3 3 1.5 33 3 3 3 3 3 3 3 3 2.0  2*  2*  2*  2*  2*  2*  2*  2*  2* 1 2.5 0 0 0 00 0 0 0 0 0 3.0 0 0 0 0 0 0 0 0 0 0 1.0 × 10⁵ 1.0 3 3 3 3 3 3 3 3 3 31.5 3 3 3 3 3 3 3 3 3 3 2.0 0  1*  1*  1*  1*  1*  1*  1*  2* 0 2.5 0 00 0 0 0 0 0 0 0 3.0 0 0 0 0 0 0 0 0 0 0 1.7 × 10⁴ 1.0 0 0 2 2 3 3 3 3 33 1.5 0 0  3*  3*  3*  3*  3*  3*  3* 2 2.0 0 0 0 0 0 0 0 0 0 0 2.5 0 00 0 0 0 0 0 0 0 3.0 0 0 0 0 0 0 0 0 0 0 2.8 × 10³ 1.0 0 0 0 0 0 3 3 3 33 1.5 0 0 0 0 0 0 0 0 0 0 2.0 0 0 0 0 0 0 0 0 0 0 2.5 0 0 0 0 0 0 0 0 00 3.0 0 0 0 0 0 0 0 0 0 0 *Indicates one or more false positives whichappears to be the result of residual enzyme activity following sporeinactivation in marginal sterilization cycles

Table IV illustrates that even with a low spore population, the sporesurvivors were predicted by enzyme activity (i.e., fluorescence) wellbefore any growth of the organism was detected.

EXAMPLE 5

This example compares read-out times for devices employing differentstrains of Bacillus stearothermophilus spores. The following strainswere tested: “ATCC 8005”, commercially available from American TypeCulture Collection, Rockville, Md.; spores obtained from growing themicroorganism contained in three different commercially availablebiological indicators, “ATTEST™ Biological Indicator”, 3M, St. Paul,Minn., “Proof PLUS Biological/Chemical Indicator”, American SterilizerCo., Erie, Pa., and “Assert Biological/Chemical Indicator”, Surgicot,Smithtown, N.Y.; “NCTC 10003” commercially available from NationCollection of Type Cultures, Colindale, London, England; “GermanEarthspore” obtained by culturing earth strips supplied by the HygieneInstitute of Hamburg, Hamburg, Germany, in tryptic soy broth afterexposure at 121° C. for 5 minutes (the 5 min. exposure was used to killall the vegetative organisms present in the earth so only B.stearothermophilus remains; and Scandinavian strain isolated from sporestrips produced by Statens Institute for Falkehelse, Oslo, Norway.

All spores were grown on a nutrient agar medium as described inExample 1. The spores were centrifuged at 11,000 rpm for 5 hours at 4°C. in density gradient commercially available as Percoll® from PharmaciaFineChemicals AB, Uppsala, Sweden. After centrifuging the spores wereresuspended in sterile distilled water. With the German Earthspore, twodistinct layers of cells were isolated in the density gradient duringcentrifugation. Using phase contact microscopy, the bottom layer wasfound to be predominantly spores and the top layer was predominantlyvegetative cells and vegetative debris with a small number of spores.Both layers were tested separately.

The spores were coated and dried on 6.35×28.58 mm (¼×⅜ inch) strips offilter paper (“S&S 591A Grade Filter Paper”) at a population of at least1×10⁶ per carrier. Devices were assembled as in Device 1, Example 1,except that in one batch of devices using the “ATCC 8005” spores, theenzyme substrate used was 4-methylumbelliferyl-beta-D-galactoside,commercially available from Sigma, 0.1 g/l dissolved in 200 μl N,N-dimethylformamide, instead of 4-methylumbelliferyl-alpha-D-glucoside,in order to detect the enzyme activity of beta-D-galactosidase on the“ATCC 8005” spores. Three unit batches were exposed at 132° C. for 1.0,1.5, 2.0, 2.5 and 3.0 minutes in an “Amsco Eagle™ Model 2013 SteamSterilizer”. After exposure the inner ampoules were crushed and theunits were incubated at 56° C. An ultraviolet light (λ=366 nm) was usedto illuminate the vials for visually read fluorescence after 10 min., 20min., 30 min., 60 min., 120 min., 180 min., and 240 minutes ofincubation. Additionally, spore growth, as indicated by a color changefrom purple to yellow, was determined visually after 24 hours ofincubation.

The results are reported in Table V.

TABLE V FLUORESCENCE Exposure Incubation Time (Minutes) at 56 C. SporeTime Number Positive/3 Tested Growth Strain (minutes) 10 20 30 40 50 60120 180 240 at 24 hr. “ATCC 8005” 1.0 3 3 3 3 3 3 3 3 3 3 (enzymesubstrate- 1.5 0 1 1 1 1 1 2 2 2 3 4-methylumbelliferyl- 2.0 0 0 0 0 0 00 0 0 0 ?-D-glucoside) 2.5 0 0 0 0 0 0 0 0 0 0 3.0 0 0 0 0 0 0 0 0 0 0“ATCC 8005” 1.0 3 3 3 3 3 3 3 3 3 3 (enzyme substrate- 1.5 0 2 3 3 3 3 33 3 3 4-methylumbelliferyl- 2.0 0 0 0 0 0 0 0 0 0 0 ?-D-galactoside) 2.50 0 0 0 0 0 0 0 0 0 3.0 0 0 0 0 0 0 0 0 0 0 “PROOF PLUS ™ 1.0 3 3 3 3 33 3 3 3 3 Biological/Chemical 1.5 3 3 3 3 3 3 3 3 3 3 Indicator” 2.0 1 33 3 3 3 3 3 3 3 2.5 0 0 0 2 3 3 3 3 3 3 3.0 0 0 0 0 0 0 0 0 0 0“ATTEST ™ 1.0 3 3 3 3 3 3 3 3 3 3 Biological 1.5 3 3 3 3 3 3 3 3 3 3Indicator” 2.0 3 3 3 3 3 3 3 3 3 3 2.5 0 0 0 0 3 3 3 3 3 3 3.0 0 0 0 0 00 0 0 0 0 “ASSERT ™ 1.0 3 3 3 3 3 3 3 3 3 3 Biological/Chemical 1.5 3 33 3 3 3 3 3 3 3 Indicator” 2.0 0 3 3 3 3 3 3 3 3 3 2.5 0 0 0 0 1 1 3 3 33 3.0 0 0 0 0 0 0 0 0 0 0 “NCTC 10003” 1.0 3 3 3 3 3 3 3 3 3 3 1.5 3 3 33 3 3 3 3 3 3 2.0 3 3 3 3 3 3 3 3 3 3 2.5 0 0 2 3 3 3 3 3 3 3 3.0 0 0 00 0 0 0 0 0 0 “German 1.0 2 3 3 3 3 3 3 3 3 3 Earthspore” 1.5 0 2 3 3 33 3 3 3 3 2.0 0 0 0 0 0 0 0 0 0 1 2.5 0 0 0 0 0 0 0 0 0 0 3.0 0 0 0 0 00 0 0 0 0 “German 1.0 0 3 3 3 3 3 3 3 3 3 Earthspore” 1.5 0 0 0 1 3 3 33 3 2 Vegatative + 2.0 0 0 0 0 0 0 0 0 1 2 Spores 2.5 0 0 0 0 0 0 0 0 00 3.0 0 0 0 0 0 0 0 0 0 0 Scandinavian 1.0 3 3 3 3 3 3 3 3 3 3 strain1.5 3 3 3 3 3 3 3 3 3 3 2.0 0 0 0 0 0  1*  2*  2*  2* 0 2.5 0 0 0 0 0 00 0 0 0 3.0 0 0 0 0 0 0 0 0 0 0 *Indicates one or more false positivereactions which appear to be the result of residual enzyme activityfollowing spore inactivation in marginal sterilization cycles

Table V illustrates that all strains of B. stearothermophilus tested hadenzyme activity (either alpha-D-glucosidase or beta-D-galactosidase)that correlated with spore survival. In most units where sporessurvived, fluorescence was detected within 2 hours of incubation and inmany units fluorescence was detected after 10 minutes of incubation. Thelayer of cells consisting mostly of vegetative cells from the GermanEarthspore also had enzyme survival following the sublethal exposures.This indicates that the alpha-D-glucosidase associated with vegetativecells could be used in this invention. Several units with theScandinavian strain had enzyme survival and no growth of the organismwith further incubation. This has been observed previously and occurs inmarginal cycles. The enzyme remains active slightly longer than thespore, and this provides an additional safety margin by monitoring moreof the cycles insuring sterility of the items in the sterilizer.

EXAMPLE 6

Bacillus stearothermophilus spores were coated on a variety of materialsto compare the fluorescent readout time. The B. stearothermophilusspores were obtained, as described in Example 1, and were suspended inethanol and deposited at approximately 1×10⁶ spores per 6.35×28.58 mm(¼×⅜ inch) strip of the following materials: polypropylene/rayonnonwoven web, commercially available as “Novonette® Nonwoven Fabric#149-190” from Kendall Fiber Products Division; nylon nonwoven web,commercially available as “Novonette® Nonwoven Fabric #149-000” fromKendall Fiber Products Division, Boston, Mass.; microporous hydrophobicfilm, commercially available as “Celgard® Microporous Hydrophobic Film2500” from Celanese Separations Products, Charlotte, N.C.; microporoushydrophilic film, commercially available as “Celgard® MicroporousHydrophilic Film 3401” from Celanese Separations Products; aluminum foilcommercially available from Reynolds, Metals Company, Richmond, Va.;filter paper, commercially available as “S&S 591A Grade Filter Paper”from Schleicher & Schuell; filter paper, commercially available as “S&S903 Grade Filter Paper” from Schleicher & Schuell; and glass fibernonwoven web, commercially available as “Manniglas #1267 Nonwoven GlassFiber Paper” from Manning Paper Company, Division of Hammermill PaperCo., Troy, N.Y. Ten microliters of the suspended spores, i.e., 1×10⁶spores, were also deposited in polypropylene vials of the samedimensions as those described in Example 1.

Devices utilizing the spore strips were assembled in accordance withExample 1, Device 1, and as illustrated in FIGS. 3 and 4, except that a1.75 mm ({fraction (11/16)} inch) in diameter piece of polypropylenenonwoven scrim, commercially available as “0.5 oz Celestra NonwovenPolypropylene” from Crown Zellerback Corp., Camas, Wash. was sandwichedbetween the spore strip 46 and the bottom of the outer vial. Thissandwich insured wetting of the spore strips with nutrient media whenampoule 48 is broken, since the scrim acts as a wick to draw nutrientmedia past the hydrophobic nylon web, microporous hydrophobic film,aluminum foil, or glass fiber nonwoven web. Devices utilizing the sporecoated vials were assembled in accordance with Example 1, Device 1,except that the device contained no spore strip or barrier. Three unitbatches of the indicators were exposed in the “Amsco Eagle™ Model 2013Steam Sterilizer” at 132° C. for 1.0, 1.5, 2.0,. 2.5 and 3.0 minutes.After exposure the ampoules containing the enzyme substrate solution andnutrient medium were crushed and the devices were incubated at 56° C.and checked for fluorescence using a longwave U.V. light (λ=366)commercially available as a “Blak-Ray® Lamp”, Model UV L-21, fromUltraviolet Products, Inc., San Gabriel, Calif., every 15 minutes for 1hour, and then hourly for up to 6 hours. Incubation was continued for 24hours, and the devices were read for growth (yellow) or no growth(purple).

The results are reported in Table VI.

TABLE VI FLUORESCENCE Exposure Incubation Time (Minutes) at 56° C. SporeCarrier Time Number Positive/3 Tested Growth Material (minutes) 15 30 4560 120 180 240 300 360 at 24 hr. Polypropylene/ 1.0 3 3 3 3 3 3 3 3 3 3rayon web 1.5 3 3 3 3 3 3 3 3 3 3 (“Novonette ® 2.0  3*  3*  3*  3*  3* 3*  3*  3*  3* 1 Nonwoven 2.5 0 0 0 0 0 0 0 0 0 0 Fabric”) 3.0 0 0 0 00 0 0 0 0 0 Microporous 1.0 3 3 3 3 3 3 3 3 3 3 hydrophobic 1.5 3 3 3 33 3 3 3 3 3 film (“Celgard ® 2.0 0 0 0 0 2 2  3*  3*  3* 2 Microporous2.5 0 0 0 0 0 0 0 0 0 0 Hydrophobic 3.0 0 0 0 0 0 0 0 0 0 0 Film 2500”)Aluminum 1.0 3 3 3 3 3 3 3 3 3 3 foil 1.5 3 3 3 3 3 3 3 3 3 3 2.0 0 0 00  1*  1*  2*  3*  3* 0 2.5 0 0 0 0 0 0 0 0 0 0 3.0 0 0 0 0 0 0 0 0 0 0Microporous 1.0 3 3 3 3 3 3 3 3 3 3 hydrophilic 1.5 3 3 3 3 3 3 3 3 3 2film (Celgard ® 2.0 0 0 0  1*  3*  3*  3*  3*  3* 0 Microporous 2.5 0 00 0 0 0 0 0 0 0 Hydrophobic 3.0 0 0 0 0 0 0 0 0 0 0 Film 3401”) Nylonnonwoven 1.0 3 3 3 3 3 3 3 3 3 3 web (“Novonette ™ 1.5 2 2 2 2 3 3 3 3 32 Nonwoven fabric” 2.0 3 3 3 3 3 3 3 3 3 3 #149-000) 2.5 0 0 0 0 0  2* 2*  2*  2* 0 3.0 0 0 0 0 0 0 0 0 0 0 Filter paper 1.0 3 3 3 3 3 3 3 3 33 (“S&S 903 1.5 3 3 3 3 3 3 3 3 3 3 Grade Filter 2.0  3*  3*  3*  3*  3* 3*  3*  3*  3* 0 Paper”) 2.5 0 0 0 0 0 0 0 0 0 0 3.0 0 0 0 0 0 0 0 0 00 Filter paper 1.0 3 3 3 3 3 3 3 3 3 3 (“S&S 591A 1.5 3 3 3 3 3 3 3 3 33 Grade Filter 2.0 0 0  1*  1*  2*  2*  2*  2*  2* 0 Paper”) 2.5 0 0 0 00 0 0 0 0 0 3.0 0 0 0 0 0 0 0 0 0 0 Glass fiber web 1.0 3 3 3 3 3 3 3 33 3 (“Manniglas 1.5 3 3 3 3 3 3 3 3 3 3 #1267 Nonwoven 2.0 1 1  2*  3* 3*  3*  3*  3*  3* 1 Glass Fiber 2.5 0 0 0 0 0 0 0 0 0 0 Paper”) 3.0 00 0 0 0 0 0 0 0 0 Polypropylene 1.0 2 3 3 3 3 3 3 3 3 3 vials 1.5 2 2 33 3 3 3 3 3 3 2.0 0 0 0 0 2 2 2 2 2 2 2.5 0 0 0 0 0 0 0 0 0 0 3.0 0 0 00 0 0 0 0 0 0 *Indicates one or more false positive reactions whichappear to be the result of residual enzyme activity following sporeinactivation in marginal sterilization cycles

In all the indicators with spores coated on carriers that survived thesteam exposure, spore survival (by enzyme activity was) detected within15 minutes. The indicators where the spores were coated directly on thevials required up to two hours to detect all instances where there wasactive alpha-D-glucosidase, and therefore spore survival. This increasedtime until reliable readout is a result of the fact that in thespore-coated vial indicators, the entire volume of medium must bemonitored for fluorescence. In contrast, with the devices which usespore strips and a barrier between the ampoule and the spore strip, onlythe spore strip is viewed for fluorescence. The barrier acts as asemi-permeable membrane to allow a small volume of media and enzymesubstrate to contact the spore carrier. Reaction of the enzyme on anyportion of the carrier with the enzyme substrate can be seen in a muchshorter time, than can reaction of enzyme with the entire contents ofthe ampoule.

EXAMPLE 7

“ATCC 9372” Bacillus subtilis was grown overnight (16 hours) at 37° C.in tryptic soy broth. This culture was used to inoculate the surface ofagar plates consisting of 8 g/l nutrient broth, 0.011 g/l manganesesulfate and 20 g/l agar at pH 7.2. The plates were incubated at 37° C.for 6 days and the spores were scraped from the plates and suspended insterile distilled water. The spores were separated from the vegetativedebris by centrifuging the suspension at 7000 rpm and 4° C. for 20minutes. The supernatant was poured off and the spores were resuspendedin sterile distilled water. This cleaning procedure was repeated severaltimes.

The Bacillus subtilis spores were coated at a population of 1.0×10⁸ on6.35×28.58 mm “S&S 591A Grade Filter Paper” strips. Devices wereassembled using these spore strips, as shown in FIGS. 3 and 4, and asdescribed in Example 1, Device 1. Three unit batches of these deviceswere preconditioned at 54° C. and 50% relative humidity for 30 minutes.The devices were then exposed for 15, 30, 60 and 120 minutes, at 54° C.and 50% relative humidity, to 600 mg/l of ethylene oxide in a Steri-Vac™400B Gas Sterilizer”, commercially available from 3M Co., St. Paul,Minn., which had been modified in accordance with the “Association forthe Advancement of Medical Instrumentation, Standard for BIER/EO GasVessels”, AAMI BEOU-3/82. After exposure, the inner ampoules wereremoved from the devices and 0.67 ml of a solution which was identicalto that contained in the inner ampoule, except that it contained 0.03g/l of 2,3,5-triphenyl tetrazolium chloride (commercially available fromICN Pharmaceuticals Inc., Cleveland Ohio), instead of bromocresol purplepH indicator dye, and 0.1 g/l 4-methylumbelliferyl-beta-D-glucoside(commercially available from Sigma), in place of the4-methylumbelliferyl-alpha-D-glucoside, was pipetted into the outervial. The devices were incubated at 37° C. An ultraviolet light (λ=366nm) was used to illuminate the devices to visually detect fluorescenceafter 30 min., 60 min., 90 min., 120 min., 180 min., 240 min. and 300min. of incubation. Additionally, spore growth, as indicated by a colorchange from colorless to red was determined visually after 24 and 168hours of incubation. The results are reported in Table VII.

TABLE VII FLUORESCENCE Ethylene Incubation Time (minutes) at 37° C.Spore Oxide Number of positives/3 tested Growth Exposure 30 60 90 120180 240 300 24 hr. 168 hr.  0 3 3 3 3 3 3 3 3 3 15 0 0 3 3 3 3 3 3 3 300 0 0 0  3*  3*  3* 0 0 60 0 0 0 0 0  3*  3* 0 0 120  0 0 0 0 0 0 0 0 0*Indicates one or more false positive reactions which appear to be theresult of residual enzyme activity following spore inactivation inmarginal sterilization cycles.

Table VII illustrates that in the devices where the spore survived the15 minute ethylene oxide exposure, the fluorescence that resulted fromthe reaction of active β-D-glucosidase with the4-methylumbelliferyl-β-D-glucoside was visually detected after 90minutes of incubation. The enzyme was completely inactivated after 120minutes of ethylene oxide exposure, demonstrating that 120 minutes ofethylene oxide exposure is a complete and efficacious sterilizationcycle. Some residual enzyme activity was detected after 3 and 4 hours ofincubation in marginal sterilization cycles of 30 and 60 minutes.

EXAMPLE 8

“ATCC 9372” Bacillus subtilis spores obtained as described in Example 7,were coated at a population of 1.0×10⁸, on 6.35×28.58 mm, “S&S 591AGrade Filter Paper” strips. Devices were assembled using these sporestrips, as shown in FIGS. 3 and 4 and as described in Example 1,Device 1. Three unit batches of the devices were preconditioned at 54°C. with 50% relative humidity for 30 minutes. The devices were thenexposed for 15, 30, 60 and 120 minutes at 54° C. and 50% relativehumidity to 600 mg/l of ethylene oxide in a “Steri-Vac™ 400B GasSterilizer”, modified as described in Example 7. After exposure thespore strips were removed from the devices and were transferred toindividual wells in a 96 well microtiter plate, commercially availableas a “Dynatech MicroFLUOR™ System” from Dynatech Laboratories, Inc.,Alexandria, Va. Each well contained 200 μl of a solution of 17 g/lbacteriological peptone, 0.17 g/l L-alanine, 0.03 g/l triphenyltetrazolium chloride and 0.1 g/l 4-methylumbelliferyl-β-D glucoside.Three negative control wells in the plate contained the solution ofnutrient medium and enzyme substrate without the spore strip. The platewas incubated at 37° C. and the fluorescence of each well was measuredafter 0, 60, 120, 180 and 240 minutes of incubation in a fluorometer,commercially available as a “3M FluoroFAST™ 96 Fluorometer” from 3MCompany, St. Paul, Minn. The results are reported in Table VIII, as anaverage of the relative fluorescence for the three wells.

TABLE VIII AVERAGE RELATIVE FLUORESCENCE (in relative fluorescenceunits) Incubation Time in (minutes) at 37° C. Exposure Time 0 60 120 180240  0 388 2329 5000 5000 5000 15 417  334  397  573 1016 30 409  321 325  373  438 60 401  309  296  299  324 120  404  312  292  292  297Negative control 278  215  195  191  186

The tests of Example 7 illustrate that Bacillus subtilis spores survivethe 15 minute ethylene oxide exposure described in this Example, and arekilled after at least a 30 minute exposure. As indicated by the wellcontaining the spore strip exposed for 15 minutes, activity of theenzyme β-D-glucosidase, and, hence, survival of the B. subtilisorganism, is indicated after 3 hours of incubation by an increase inrelative fluorescence of at least 2.5 times the background levelfluorescence of the negative control after 3 hours of incubation.Likewise, in the unexposed control, an increase of relative fluorescencegreater than 2.5 times the background level fluorescence of the negativecontrol, after only 1 hour of incubation, indicates enzyme activity andspore survival.

However, the strips exposed to ethylene oxide sterilization for at least30 minutes, had relative fluorescence values of less than 2.5 times thenegative control after 3 hours of incubation. Thus, indicating onlyresidual enzyme activity and the lack of spore survival.

FIG. 7 is the graphic representation of the results reported in TableVIII. In the figure line A represents the device exposed for 15 minutes,line B represents the device exposed for 30 minutes, line C representsthe device exposed for 60 minutes, line D represents the device exposedfor 120 minutes and line E represents the negative control.

EXAMPLE 9

“ATCC 9372” Bacillus subtilis spores, obtained as described in Example7, were coated at a population of 1.0×10⁸ on 6.35×28.58 mm (¼×⅜ inch)strips of “S&S 591A Grade Filter Paper”. Devices were assembled usingthese spore strips, as shown in FIGS. 3 and 4 and as described inExample 1, Device 1. Three unit batches of the devices werepreconditioned at 54° C. with 50% relative humidity for 30 minutes. Thedevices were then exposed for 15, 30, 60 and 120 minutes, at 54° C. and50% relative humidity, to 600 mg/l of ethylene oxide in a “Steri-Vac™400B Gas Sterilizer”, modified as described in Example 7. After exposurethe spore strips, along with three identical unexposed spore strips wereremoved from the devices and were transferred to individual wells in a96 well microtiter plate, commercially available as a “DynatechMicroFLUOR™ System” from Dynatech Laboratories, Inc., Alexandria, Va.Each well contained 200 μl of a solution of 17 g/l bacteriologicalpeptone, 0.17 g/l L-alanine, 0.03 g/l triphenyl tetrazolium chloride and0.1 g/l methylumbelliferyl-alpha-D-arabinofuranoside, commerciallyavailable from Sigma. Three negative control wells in the platecontained the solution of nutrient medium and enzyme substrate withoutthe spore strip. The plate was incubated at 37° C. and the fluorescenceof each well was measured after 0, 60, 120, 180 and 240 minutes ofincubation in a fluorometer, commercially available as a “3M FluoroFAST™96 Fluorometer” from 3M Company, St. Paul, Minn. The results arerecorded, as an average of the relative fluorescence for the three wellsin FIG. 8, wherein average relative fluorescence is plotted againstincubation time for wells containing spores exposed for 15, 30, 60 and120 minutes, and the negative control. In the figure, F represents thedevice exposed for 15 minutes, G represents the device exposed for 30minutes, H represents the device exposed for 60 minutes, J representsthe device exposed for 120 minutes, and K represents the negativecontrol.

The results of Example 7 illustrate that B. subtilis spores survive a 15minute ethylene oxide exposure of the type described above, and arekilled after at least a 30 minute exposure. In this example thealpha-D-arabinofuranosidase still had residual activity after the 120minute cycle, which is considered a complete and efficacioussterilization cycle. This level of fluorescence is considered thebackground level, and any significant increase above this levelindicates a sterilization failure. For example, after 3 hours ofincubation, the difference in relative fluorescence units (RFU) betweenthe strip exposed for 15 minutes and the strip exposed for 120 minutesis 2166. This difference is significant and indicates that 15 minutes isan incomplete sterilization cycle for the ethylene oxide conditionsutilized. However, the difference between the strip exposed for 30minutes and the strip exposed for 120 minutes is only 745 RFU. Thisdifference is considered insignificant and indicates enzyme residualactivity in marginal sterilization cycles of 30 minutes.

EXAMPLE 10

Bacillus stearothermophilus spores (“ATCC 7953”), obtained as describedin Example 1, were suspended in distilled water, after one wash, at apopulation of 1×10⁸ spores/ml. The following procedure was used topurify the enzyme alpha-D-glucosidase. The suspension (200 ml) wasdialyzed against 2 l of a solution of 10 mM acetate buffer and 5 mMCaCl₂, pH 6.2, overnight at 4° C. Insoluble residues were then removedby centrifugation. The supernatant was fractionated with solid ammoniumsulfate. The precipitates from 20%-60% ammonium sulfate were collectedon a Buchner funnel containing a filter pad. The filter pad was preparedby passing a suspension (100 g/l) of “Celite® Filter Aid”, commerciallyavailable from Manville Specialty Product Group, Lompoc, Calif. over twosheets of Whatman No. 1 filter paper. After filtration, the “Celite®Filter Aid” pad was suspended in 20 ml of the solution of 10 mM acetatebuffer and 5 mM CaCl₂, pH 6.2, and stirred for 4 hours at 4° C. “Celite®Filter Aid” was removed from the soluble enzyme by filtration. The lightbrown enzyme solution was dialyzed overnight against 4 L of the solutionof 10 mM acetate buffer and 5 mM CaCl₂ at 4° C. The dialyzed solutionwas removed from the dialysis tubing and filtered to remove insolubledebris.

The dialyzed solution was adjusted to pH 6.2 and two volumes of coldacetone (−20° C.) were added with stirring. The acetone-enzyme solutionwas held at −20° C. for 6 hours. The light brown precipitate wascollected by gravity filtration at 4° C. and dissolved in 10 ml of thesolution of 10 mM acetate buffer and 5 mM CaCl₂, pH 6.2. The light ambersolution was adjusted to pH 5.5 by the addition of 0.1 M acetate buffer,pH 4.6. The solution was again treated with two volumes of cold acetone(−20° C.) and stored at 20° C. overnight. The precipitate was collectedby gravity filtration and dissolved in 10 ml of the solution of 10 mMacetate buffer and 5 mM CaCl₂, pH 6.2. The light amber solution wasdialyzed for 48 hours against 4 L of a solution of 50 mM phosphatebuffer and 5 mM EDTA, pH 6.2 (Buffer A), at 4° C. with complete bufferchange every 24 hours.

Five ml of the dialyzed sample was loaded in a column (2.5×30 cm),packed with “DEAE-Sephadex® Beads”, commercially available fromPharmacia, Inc., Piscataway, N.J., and equilibrated with 50 mM phosphatebuffer and 5 mM EDTA, pH 6.2. The column was washed successively with:a) 500 ml of Buffer A, and b) 200 ml of a linear 0-0.8 M NaCl gradientin Buffer A. The flow rate was kept at approximately 5 ml/60 min. Theactive fractions that appeared were collected and dialyzed for 48 hoursagainst 4 L of distilled water, with a complete change after 24 hours.The lyopholized fractions were suspended in a 3 ml of a solution of 150mM phosphate buffer and 5 mM EDTA, pH 6.2 (Buffer B). This suspensionwas passed through a column (2.5×30 cm) packed with “Sephadex® G-100Beads”, commercially available from Pharmacia, Inc., with Buffer B at anapproximate flow rate of 15 ml/60 min. The active fractions werecollected and dialyzed against 4 L of distilled water. The dialyzedfractions were then lyopholized.

Seven 6.35×28.58 (¼×⅜ inch) strips of “S&S 903 Grade Filter Paper” weresaturated with a suspension of 0.02 M purified alpha-D-glucosidase indistilled water. Seven other paper strips, of the same type anddimension, were saturated with a 1×10⁶ spores/ml solution of B.stearothermophilus spores, (“ATCC 7953”), obtained as described inExample 1, suspended in distilled water. All carrier strips were airdried overnight at ambient temperature (20° C.).

Devices were assembled using these spore strips as shown in FIGS. 3 and4, and described in Example 1 as Device 1. These devices were exposed ina gravity displacement steam sterilizer, commercially available as an“Amsco Eagle™ Model 2013 Sterilizer” at 132° C. and 469.4 kg/cm² (33psi) for 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 minutes. After exposure theinner ampoules containing the enzyme substrate and nutrient medium werecrushed and the units were incubated at 56° C. for 24 hours. Anultraviolet light (λ=366 nm) was used to illuminate the vials forvisually read fluorescence.

Relative fluorescence of each device was also measured using a 3MFluoroFAST™ 96 Fluorometer”, at 366 nm. The results are recorded inTable IX.

TABLE IX FLUORESCENCE Device with Device with Exposure purified enzymespore strip Time Fluorometer Fluorometer (min) Observed¹ (RFU) Observed¹(RFU) 0.0 + 3954 + 3182 0.5 + 1421 + 1876 1.0 +  860 + 1350 1.5 +  513 + 830 2.0 +  456 +  636 2.5 −  367 −  357 3.0 −  322 −  312 ¹“+”indicates fluorescence by visual observation while “−” indicates nofluorescence by visual observance.

The results in Table IX illustrate that the devices which employ thepurified enzyme had the same visually observed fluorescence, andapproximately the same measured fluorescence, as the devices employingB. stearothermophilus. Thus, this example illustrates that activity ofthe purified enzyme alone, not bound to the spore from which it isderived, is useful to detect spore survival.

What is claimed is:
 1. A method of detecting the presence of viablemicroorganisms after a sterilization cycle comprising the steps of: (a)exposing a source of active enzyme to a sterilization cycle; (b)contacting the active enzyme after the sterilization cycle with anaqueous buffer and a substrate which is specific for and reacts with theactive enzyme at about 20-70° C. to form an enzyme modified product; (c)adding a color developer which is specific for and reacts with theenzyme modified product to generate a color in the presence of viablemicroorganisms at about 20-70° C.; (d) determining the existence of saidcolor by visual means in about ten minutes to about 60 minutes; and (e)correlating the existence of said color with the presence of viablemicroorganisms.
 2. The method of claim 1 wherein the viablemicroorganisms are bacterial spores.
 3. The method of claim 2 whereinthe viable microorganisms are selected from the group consisting ofBacillus stearothermophilus spores and Bacillus subtilis spores.
 4. Themethod of claim 1 wherein the substrate is selected from the groupconsisting of: 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside;4-methylumbelliferyl-a-D-glucoside; L-tyrosine-7-amido-4-methylcoumarin;L-leucine-7-amido-4-methylcoumarin; L-alanine-7-amido-4-methylcoumarin;L-phenylalanine-7-amido-4-methylcoumarin;L-proline-7-amido-4-methylcoumarin; L-leucyl-2-naphthylamide;L-valyl-2-naphthylamide; and L-cystyl-2-naphthylamide.
 5. The method ofclaim 1 wherein the sterilization cycle uses a sterilization meansselected from the group consisting of saturated steam, dry heat,radiation, and ethylene oxide.
 6. The method of claim 1 wherein steps(b) and (c) are performed between about 20° C. and about 30° C.
 7. Themethod of claim 1 wherein the aqueous buffer consists of tris(hydroxymethyl)(aminomethane HCl) in water at a pH of 7.5-9.5.
 8. Themethod of claim 1 wherein the source of the viable microorganisms isimpregnated onto a filter.
 9. The method of claim 1 wherein the sourceof active enzyme is the viable microorganisms.
 10. In a method ofdetecting the presence of viable microorganisms after the completion ofa sterilization cycle, wherein a source of active enzyme is exposed tothe sterilization cycle, is contacted with a substrate specific for theactive enzyme after the sterilization cycle to form an enzyme modifiedproduct, and then is contacted with a means for detecting the enzymemodified product as an indication of the presence of viablemicroorganisms, the improvement comprising: (I) using an aqueous bufferand a substrate which forms a complex with said active enzyme at 20-70°C.; (II) using a color developer which reacts with the enzyme modifiedproduct to generate a color at 20-70° C. as the means of detecting theproduct, determining the existence of said color by visual means inabout 10 minutes to about 60 minutes; and (III) correlating theexistence of said color with the presence of viable microorganisms. 11.An indicator for detecting the presence of viable microorganisms after asterilization cycle comprising: (a) a filter containing a source ofactive enzyme; (b) an aqueous buffer; (c) a substrate specific for theactive enzyme, which substrate reacts with the active enzyme at 20-70°C.; and (d) a color developer specific for the substrate, whichdeveloper reacts with the substrate in the presence of the active enzymeto generate a color at 20-70° C. which can be detected by visual means.